Binding proteins 2

ABSTRACT

The present disclosure relates to cell penetrating anti-DNA binding proteins. Compositions comprising these binding proteins may be may be useful for delivering agents to cells and treating diseases such as cancer.

FIELD OF THE INVENTION

The present disclosure relates to cell penetrating anti-DNA bindingproteins. Compositions comprising these binding proteins may be usefulfor delivering agents to cells and treating diseases such as cancer.

BACKGROUND OF THE INVENTION

Development of cell penetrating anti-DNA binding proteins as therapeuticagents for human diseases has great clinical potential, in particularbecause of their ability to selectively impair DNA repair pathwaysand/or deliver various therapeutic payloads to target cells.

Accordingly, improved cell penetrating anti-DNA binding proteins arerequired.

SUMMARY OF THE INVENTION

The present inventors have identified cell penetrating anti-DNA bindingprotein modifications that surprisingly increase nuclear penetration. Insome cases, these modifications may also improve physical stability andreduce immunogenicity.

Accordingly, in a first example, the present disclosure relates to acell penetrating anti-DNA binding protein having an antigen bindingdomain, wherein the antigen binding domain binds to or specificallybinds to DNA and comprises a heavy chain variable region (V_(H)) havinga complementarity determining region (CDR) 1 as shown in SEQ ID NO: 1, aCDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3 and a CDR3 as shown in SEQID NO: 4 and a light chain variable region (V_(L)) having a CDR1 asshown in SEQ ID NO: 5 or SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7and a CDR3 as shown in SEQ ID NO: 8. In this example, the CDRs have beendefined using Kabat.

In another example, the present disclosure relates to a cell penetratinganti-DNA binding protein having an antigen binding domain, wherein theantigen binding domain binds to or specifically binds to DNA andcomprises:

a heavy chain variable region (V_(H)) having a complementaritydetermining region (CDR) 1 as shown in SEQ ID NO: 9, a CDR2 as shown inSEQ ID NO: 10 or SEQ ID NO: 11 and a CDR3 as shown in SEQ ID NO: 12;

a light chain variable region (V_(L)) having a CDR1 as shown in SEQ IDNO: 13 or SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 15 and a CDR3 asshown in SEQ ID NO: 16. In this example, the CDRs have been definedusing IMGT.

In another example, binding proteins according to the present disclosurecomprise:

(i) a V_(H) comprising a sequence at least 95% identical to the sequenceas shown in any one of SEQ ID NOs: 17 to 23;

(ii) a V_(L) comprising a sequence at least 95% identical to thesequence as shown in any one of SEQ ID NOs: 24 to 29; or

(iii) a V_(H) comprising a sequence at least 95% identical to thesequence as shown in any one of SEQ ID NOs: 17 to 23 and a V_(L)comprising a sequence at least 95% identical to the sequence as shown inany one of SEQ ID NOs: 24 to 29. For example, the binding protein maycomprise a sequence at least 95% identical to the sequence as shown inany one of SEQ ID NOs: 17 to 23. In another example, the binding proteinmay comprise a V_(L) comprising a sequence at least 95% identical to thesequence as shown in any one of SEQ ID NOs: 24 to 29. In another example,the binding protein may comprise a V_(H) comprising a sequence at least95% identical to the sequence as shown in any one of SEQ ID NOs: 17 to23 and a V_(L) comprising a sequence at least 95% identical to thesequence as shown in any one of SEQ ID NOs: 24 to 29.

In another example, the V_(H) and a V_(L) are separated by a linker. Forexample, the linker may be comprise (Gly₄Ser)₃. In another example thelinker comprises an amino acid sequence as shown in SEQ ID NO: 30.

In an example, the V_(H) and V_(L) are in a single polypeptide chain.For example, the binding protein may be:

(i) a single chain Fv fragment (scFv);

(ii) a dimeric scFv (di-scFv);

(iii) a trimeric scFv (tri-scFv);

(iv) any one of (i), (ii) or (iii) linked to a constant region of anantibody, Fc or a heavy chain constant domain C_(H)2 and/or C_(H)3. Forexample, the binding protein may be a di-scFv. In this example, thescFv's may be separated by a linker. For example, the linker maycomprise an amino acid sequence as shown in SEQ ID NO: 31.

In another example, the V_(H) and V_(L) are in separate polypeptidechains. For example, the binding protein may be:

(i) a diabody;

(ii) a triabody;

(iii) a tetrabody;

(iv) a Fab;

(v) a F(ab′)₂;

(vi) a Fv;

(vii) one of (i) to (vi) linked to a constant region of an antibody, Fcor a heavy chain constant domain C_(H)2 and/or C_(H)3; or,

(viii) an intact antibody.

Thus, the V_(H) and V_(L) of an Fv can be formed of a single peptidechain (e.g. scFv), or can be formed of two separate peptide chains.

In an example, the binding protein is humanized.

In another example, the present disclosure relates to a cell penetratinganti-DNA Fv fragment having an antigen binding domain, wherein theantigen binding domain binds to or specifically binds to DNA andcomprises at least one of:

a V_(H) having a CDR 1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQID NO: 2 or SEQ ID NO: 3, a CDR3 as shown in SEQ ID NO: 4 and a V_(L)having a CDR1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6, a CDR2 as shownin SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8;

a V_(H) having a CDR 1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQID NO: 10 or SEQ ID NO: 11, a CDR3 as shown in SEQ ID NO: 12 and a V_(L)having a CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, a CDR2 asshown in SEQ ID NO: 15 and a CDR3 as shown in SEQ ID NO: 16;

a V_(H) comprising a sequence at least 95% identical to the sequence asshown in any one of SEQ ID NOs: 17 to 23 and a V_(L) comprising asequence at least 95% identical to the sequence as shown in any one ofSEQ ID NOs: 24 to 29.

In this example, the Fv fragment may be a di-scFv. In an example, the Fvfragment may comprise an amino acid sequence as shown in any one of SEQID NOs: 32-47. For example, the Fv fragment may comprise an amino acidsequence as shown in SEQ ID NO: 41.

In some embodiments, the Fv is naked. In another example, the Fvfragment may be conjugated to another compound.

In an example, the Fv is humanized. For example, the Fv may be ahumanized di-scFv.

In another example, the present disclosure relates to a nucleic acidsequence encoding an above referenced binding proteins. Exemplarynucleic acid sequences are shown in SEQ ID NOs: 51-66. The disclosednucleic acid sequences can be codon-optimized to increase levels ofexpression for synthesizing the proteins. In another example, thepresent disclosure relates to an expression vector comprising a nucleicacid sequence according to the present disclosure. For example, theexpression vector may comprise a nucleic acid sequences are shown in anyone of SEQ ID NOs: 51-66 or a codon optimized sequence thereof.

In another example, the present disclosure relates to a host cellcomprising an above referenced binding protein, nucleic acid or vector,or codon optimized sequence thereof.

In another example, the present disclosure relates to a method oftreating cancer. For example, a method of treating cancer comprisingadministering to a subject an Fv fragment comprising an amino acidsequence as shown in any one of SEQ ID NOs: 32, 36, 41 or 43. Forexample, an Fv fragment comprising an amino acid sequence as shown inSEQ ID NOs: 32 may be administered to a subject. In another example, anFv fragment comprising an amino acid sequence as shown in SEQ ID NOs: 36may be administered to a subject. In another example, an Fv fragmentcomprising an amino acid sequence as shown in SEQ ID NOs: 41 may beadministered to a subject. In another example, an Fv fragment comprisingan amino acid sequence as shown in SEQ ID NOs: 43 may be administered toa subject. In an example, the cancer is colon cancer, brain cancer,prostate cancer, ovarian cancer, endometrial cancer, breast cancer, orpancreatic cancer. For example, the cancer may be colon cancer or braincancer. In an example, the cancer is brain cancer. In an example, thebrain cancer is glioblastoma.

In another example, the present disclosure relates to use of a bindingprotein such as an Fv fragment, composition, vector or host cellaccording to the present disclosure in the manufacture of a medicamentfor treating cancer. In another example, the present disclosure relatesto a binding protein such as an Fv fragment, composition, vector or hostcell according to the present disclosure for use in treating cancer.

The experimental results below also illustrate that binding proteinsdisclosed herein can work with poly (ADP-ribose) polymerase (PARP)inhibitors to kill cancer cells. Accordingly, in another example, thepresent disclosure relates to a method of treating cancer in a subjectin need thereof, the method comprising administering to the subject abinding protein or Fv fragment defined herein and a PARP inhibitor.

In an example, the PARP inhibitor is olaparib.

In an example, the cancer is substantially HDR deficient. In anotherexample, the cancer is substantially BRCA2 deficient. In anotherexample, the cancer is substantially PTEN deficient. In an example, thecancer is colon cancer, brain cancer, prostate cancer, ovarian cancer,endometrial cancer, breast cancer, or pancreatic cancer. For example,the cancer may be colon cancer or brain cancer. In an example, thecancer is brain cancer. In an example, the brain cancer is glioblastoma.In an example, the cancer is resistant to PARP inhibition. For example,the cancer may be resistant to treatment with olaparib. In anotherexample, the cancer is triple negative breast cancer.

In another example, the present disclosure relates to a therapeuticcombination comprising a binding protein or Fv fragment defined hereinand a PARP inhibitor, the combination being provided for simultaneous orsequential administration. In another example, the present disclosurerelates to a therapeutic combination comprising:

a binding protein or Fv comprising the CDRs of SEQ ID NOs: 41; or,

a binding protein comprising the amino acid sequence shown in SEQ ID NO:41;

and, a PARP inhibitor, the combination being provided for simultaneousor sequential administration. For example, the binding protein or Fv cancomprise heavy chain CDRs as shown in SEQ ID NOs: 1, 3 and 4 and lightchain CDRs as shown in SEQ ID NOs: 6, 7 and 8. In these examples, thetherapeutic combination may be used for treating cancer. Furthermore, inthese examples, the PARP inhibitor may be olaparib.

Any example herein shall be taken to apply mutatis mutandis to any otherexample unless specifically stated otherwise.

The present disclosure is not to be limited in scope by the specificexamples described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the disclosure, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The disclosure is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. Images illustrating the results of SDS-PAGE analysis of reducedand denatured variants.

FIG. 2. Images illustrating the results of SDS-PAGE analysis ofnon-reduced variants.

FIG. 3. Images illustrating the results of Alkaline phosphatase-basedsurvey of nuclear penetration.

FIG. 4. Plots showing Quantitative analysis of the alkalinephosphatase-based survey of nuclear penetration.

FIG. 5. Histograms of Quantitative analysis of the alkalinephosphatase-based survey of nuclear penetration.

FIG. 6. Images illustrating the results of Immunofluorescence-basedsurvey of nuclear penetration.

FIG. 7. Plots showing Quantitative analysis of theimmunofluorescence-based survey of nuclear penetration.

FIG. 8, Panel A. Exemplary images showing Accumulation of DNA damage inPTEN-proficient and PTEN-deficient cancer cells.

FIG. 8, Panel B. Histogram showing Accumulation of DNA damage inPTEN-proficient and PTEN-deficient cancer cells.

FIG. 9. Histogram showing Cell viability of PTEN-deficient cancer cells.

FIG. 10, Panel A. Exemplary images showing Accumulation of DNA damage inBRCA2-proficient and BRCA2-deficient cancer cells.

FIG. 10, Panel B. Histogram showing Accumulation of DNA damage inBRCA2-proficient and BRCA2-deficient cancer cells.

FIG. 11. di-scFv (SEQ ID NO: 41) penetrates HDR deficient DLD-1 coloncancer cell nuclei.

FIG. 12. di-scFv (SEQ ID NO: 41) penetrates HDR deficient MCF-7 breastcancer cell nuclei.

FIG. 13. More than additive cell death mediated by di-scFv (SEQ ID NO:41) and PARP inhibitor in HDR deficient cancer cells (*p<0.05 comparedto olaparib or the di-scFv alone).

FIG. 14. di-scFv (SEQ ID NO: 41) kills primary human glioblastoma cells.

FIG. 15. di-scFv (SEQ ID NO: 41) penetrates human glioblastoma spheres(A) and reduces sphere volume in a time-dependent (B) and dose-dependent(C) manner.

FIG. 16. In-vivo assessment of di-scFv (SEQ ID NO: 41) in a orthotopicmouse model of glioblastoma. a) Representative H&E stained brainsections from mice treated with control or di-scFv (SEQ ID NO: 41) andcorresponding quantification of area. (*P<0.04, n=3). b) Representativemicrographs of TUNEL staining, and corresponding percentage ofTUNEL-positive cells. * denotes a P≤0.05 as determined by a one-wayANOVA test. c) Protein L-based immunostaining for comparison of di-scFv(SEQ ID NO: 41) in tumour and adjacent brain tissue. d) Body weight(grams) profile for mice in the survival study (n=7). e) Survival datafor control PBS versus di-scFv (SEQ ID NO: 41) treatment arms (n=7,P=0.02, Mantel-Cox test).

FIG. 17. Effect of di-scFv (SEQ ID NO: 41) on Foci Accumulation. Thepercentage of P53BP1-positive cells increased in HDR-deficient DLD1 andU251 cells following 24 hour PAT-DX1 and combination treatment(s).

KEY TO SEQUENCE LISTING

-   SEQ ID NO: 1—Heavy Chain CDR1 KABAT-   SEQ ID NO: 2—Heavy Chain CDR2 (variants 2-4, 6-8, 10-12) KABAT-   SEQ ID NO: 3—Heavy Chain CDR2 (variants 13-19) KABAT-   SEQ ID NO: 4—Heavy Chain CDR3 KABAT-   SEQ ID NO: 5—Light Chain CDR1 (variants 2-4, 6-8, 10-12) KABAT-   SEQ ID NO: 6—Light Chain CDR1 (variants 13-19) KABAT-   SEQ ID NO: 7—Light Chain CDR2 KABAT-   SEQ ID NO: 8—Light Chain CDR3 KABAT-   SEQ ID NO: 9—Heavy Chain CDR1 IMGT-   SEQ ID NO: 10—Heavy Chain CDR2 (variants 2-4, 6-8, 10-12) IMGT-   SEQ ID NO: 11—Heavy Chain CDR2 (variants 13-19) IMGT-   SEQ ID NO: 12—Heavy Chain CDR3 IMGT-   SEQ ID NO: 13—Light Chain CDR1 (variants 2-4, 6-8, 10-12) IMGT-   SEQ ID NO: 14—Light Chain CDR1 (variants 13-19) IMGT-   SEQ ID NO: 15—Light Chain CDR2 IMGT-   SEQ ID NO: 16—Light Chain CDR3 IMGT-   SEQ ID NO: 17—Heavy Chain variable region (variants 2, 6 and 10)-   SEQ ID NO: 18—Heavy Chain variable region (variants 3, 7 and 11)-   SEQ ID NO: 19—Heavy Chain variable region (variants 4, 8 and 12)-   SEQ ID NO: 20—Heavy Chain variable region (variants 6 and 10)-   SEQ ID NO: 21—Heavy Chain variable region (variants 13, 16 and 19)-   SEQ ID NO: 22—Heavy Chain variable region (variants 14 and 17)-   SEQ ID NO: 23—Heavy Chain variable region (variants 15 and 18)-   SEQ ID NO: 24—Light Chain variable region (variants 2, 3 and 4)-   SEQ ID NO: 25—Light Chain variable region (variants 6, 7 and 8)-   SEQ ID NO: 26—Light Chain variable region (variants 10, 11 and 12)-   SEQ ID NO: 27—Light Chain variable region (variants 13, 14 and 15)-   SEQ ID NO: 28—Light Chain variable region (variants 16, 17 and 18)-   SEQ ID NO: 29—Light Chain variable region (variant 19)-   SEQ ID NO: 30—Linker sequence 1-   SEQ ID NO: 31—Linker sequence 2-   SEQ ID NO: 32—Variant 2-   SEQ ID NO: 33—Variant 3-   SEQ ID NO: 34—Variant 4-   SEQ ID NO: 35—Variant 6-   SEQ ID NO: 36—Variant 7-   SEQ ID NO: 37—Variant 8-   SEQ ID NO: 38—Variant 10-   SEQ ID NO: 39—Variant 11-   SEQ ID NO: 40—Variant 12-   SEQ ID NO: 41—Variant 13-   SEQ ID NO: 42—Variant 14-   SEQ ID NO: 43—Variant 15-   SEQ ID NO: 44—Variant 16-   SEQ ID NO: 45—Variant 17-   SEQ ID NO: 46—Variant 18-   SEQ ID NO: 47—Variant 19-   SEQ ID NO: 48—Heavy Chain variable region murine (D31N) anti-DNA    binding antibody-   SEQ ID NO: 49—Light Chain variable region murine (D31N) anti-DNA    binding antibody-   SEQ ID NO: 50—(D31N) murine prototype produced from P. pastoris-   SEQ ID NO: 51—DNA sequence Variant 2-   SEQ ID NO: 52—DNA sequence Variant 3-   SEQ ID NO: 53—DNA sequence Variant 4-   SEQ ID NO: 54—DNA sequence Variant 6-   SEQ ID NO: 55—DNA sequence Variant 7-   SEQ ID NO: 56—DNA sequence Variant 8-   SEQ ID NO: 57—DNA sequence Variant 10-   SEQ ID NO: 58—DNA sequence Variant 11-   SEQ ID NO: 59—DNA sequence Variant 12-   SEQ ID NO: 60—DNA sequence Variant 13-   SEQ ID NO: 61—DNA sequence Variant 14-   SEQ ID NO: 62—DNA sequence Variant 15-   SEQ ID NO: 63—DNA sequence Variant 16-   SEQ ID NO: 64—DNA sequence Variant 17-   SEQ ID NO: 65—DNA sequence Variant 18-   SEQ ID NO: 66—DNA sequence Variant 19-   SEQ ID NO: 67—(GGGGS)₃ linker-   SEQ ID NO: 68—3E10 human IgG1 L2345A/L235A heavy chain full length    sequence-   SEQ ID NO: 69—3E10 human IgG1 constant heavy region 1-   SEQ ID NO: 70—3E10 human IgG1 hinge region-   SEQ ID NO: 71—3E10 human IgG1 L2345A/L235A constant heavy region 2-   SEQ ID NO: 72—3E10 human IgG1 constant heavy region 3-   SEQ ID NO: 73—3E10 human IgG1 N297D heavy chain full length sequence-   SEQ ID NO: 74—3E10 human IgG1 N297D constant heavy region 2-   SEQ ID NO: 75—3E10 human IgG1 L2345A/L235A/N297D heavy chain full    length sequence-   SEQ ID NO: 76—3E10 human IgG1 L2345A/L235A/N297D constant heavy    region 2-   SEQ ID NO: 77—Unmodified constant heavy region 2-   SEQ ID NO: 78—Light chain full length sequence

DETAILED DESCRIPTION OF THE INVENTION General Techniques and SelectedDefinitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., molecular biology,biochemistry, antibodies, antibody fragments such as single chainfragment variable and clinical studies).

The term “cell-penetrating” is used in the context of the presentdisclosure to refer to an anti-DNA binding protein such as an antigenbinding fragment that is transported into the nucleus of livingmammalian cells and binds DNA (e.g., single-stranded and/ordouble-stranded DNA). In an example, a cell-penetrating anti-DNA bindingprotein is transported into the nucleus of a cell without the aid of acarrier or conjugate.

The term “anti-DNA binding protein” is used in the context of thepresent disclosure to refer to proteins capable of binding DNA.Exemplary binding proteins include immunoglobulin, antibodies andantigenic binding fragments. Other examples of binding proteins arediscussed below.

The term “immunoglobulin” will be understood to include any anti-DNAbinding protein comprising an immunoglobulin domain. Exemplaryimmunoglobulins are antibodies. Additional proteins encompassed by theterm “immunoglobulin” include domain antibodies, camelid antibodies andantibodies from cartilaginous fish (i.e., immunoglobulin new antigenreceptors (IgNARs)). Generally, camelid antibodies and IgNARs comprise aV_(H), however lack a V_(L) and are often referred to as heavy chainimmunoglobulins. Other “immunoglobulins” include T cell receptors.

The term “antibody” is used in the context of the present disclosure torefer to immunoglobulin molecules immunologically reactive with aparticular antigen and includes both polyclonal and monoclonalantibodies. The term also includes genetically engineered forms such aschimeric antibodies (e.g., humanized murine antibodies) andheteroconjugate antibodies (e.g., bispecific antibodies). The term“antibody” also includes antigen binding forms of antibodies, includingfragments with antigen-binding capability (e.g., Fab′, F(ab′)₂, Fab, Fvand rIgG as discussed in Pierce Catalogue and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed.,W.H. Freeman & Co., New York (1998). The term is also used to refer torecombinant single chain Fv fragments (scFv) as well as divalent(di-scFv) and trivalent (tri-scFV) forms thereof. The term antibody alsoincludes bivalent or bispecific molecules, diabodies, triabodies, andtetrabodies. Examples of bivalent and bispecific molecules are describedin Kostelny et al. (1992) J Immunol 148:1547; Pack and Pluckthun (1992)Biochemistry 31:1579; Hollinger et al., 1993, supra, Gruber et al.(1994) J. Immunol.:5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al.(1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, andMcCartney, et al. (1995) Protein Eng. 8:301.

An “antigen binding fragment” of an antibody comprises one or morevariable regions of an intact antibody. Examples of antibody fragmentsinclude Fab, Fab′, F(ab′)₂ and Fv fragments; diabodies; linearantibodies; single-chain antibody molecules and multispecific antibodiesformed from antibody fragments. For example, the term antigen bindingfragment may be used to refer to recombinant single chain Fv fragments(scFv) as well as divalent (di-scFv) and trivalent (tri-scFV) formsthereof. Such fragments can be produced via various methods known in theart. For example, di-scFv encompassed by the present disclosure can beproduced and purified by the methods described in Example 1 below.

The terms “full-length antibody”, “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antigen binding fragment of an antibody.Specifically, whole antibodies include those with heavy and light chainsincluding an Fc region. The constant domains may be wild-type sequenceconstant domains (e.g., human wild-type sequence constant domains) oramino acid sequence variants thereof.

As used herein, “variable region” refers to the portions of the lightand/or heavy chains of an antibody as defined herein that specificallybinds to an antigen and, for example, includes amino acid sequences ofCDRs; i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). Forexample, the variable region comprises three or four FRs (e.g., FR1,FR2, FR3 and optionally FR4) together with three CDRs. V_(H) refers tothe variable region of the heavy chain. V_(L) refers to the variableregion of the light chain.

As used herein, the term “complementarity determining regions” (syn.CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues ofan antibody variable region the presence of which are major contributorsto specific antigen binding. Each variable region typically has threeCDR regions identified as CDR1, CDR2 and CDR3. In one example, the aminoacid positions assigned to CDRs and FRs are defined according to KabatSequences of Proteins of Immunological Interest, National Institutes ofHealth, Bethesda, Md., 1987 and 1991 (also referred to herein as “theKabat numbering system” or “Kabat”.

Other conventions that include corrections or alternate numberingsystems for variable domains include IMGT (Lefranc, et al. (2003), DevComp Immunol 27: 55-77), Chothia (Chothia C, Lesk A M (1987), J Mal Biol196: 901-917; Chothia, et al. (1989), Nature 342: 877-883) and AHo(Honegger A, Plückthun A (2001) J Mol Biol 309: 657-670). Forconvenience, examples of binding proteins of the present disclosure mayalso be labelled according to IMGT. These examples are expresslyindicated as such. For example, see SEQ ID NO: 9-16.

“Framework regions” (Syn. FR) are those variable domain residues otherthan the CDR residues.

The term “constant region” as used herein, refers to a portion of heavychain or light chain of an antibody other than the variable region. In aheavy chain, the constant region generally comprises a plurality ofconstant domains and a hinge region, e.g., a IgG constant regioncomprises the following linked components, a constant heavy C_(H)1, alinker, a C_(H)2 and a C_(H)3. In a heavy chain, a constant regioncomprises a Fc. In a light chain, a constant region generally compriseone constant domain (a CL1).

The term “fragment crystalizable” or “Fc” or “Fc region” or “Fc portion”(which can be used interchangeably herein) refers to a region of anantibody comprising at least one constant domain and which is generally(though not necessarily) glycosylated and which is capable of binding toone or more Fc receptors and/or components of the complement cascade.The heavy chain constant region can be selected from any of the fiveisotypes: α, δ, ε, γ, or μ. Exemplary heavy chain constant regions aregamma 1 (IgG1), gamma 2 (IgG2) and gamma 3 (IgG3), or hybrids thereof.

A “constant domain” is a domain in an antibody the sequence of which ishighly similar in antibodies/antibodies of the same type, e.g., IgG orIgM or IgE. A constant region of an antibody generally comprises aplurality of constant domains, e.g., the constant region of γ, α or δheavy chain comprises two constant domains.

The term “naked” is used to refer to binding proteins of the presentdisclosure that are not conjugated to another compound, e.g., a toxiccompound or radiolabel. For example, the term “naked” can be used torefer to binding proteins such as di-scFv that are not conjugated toanother compound. Accordingly, in one example, the binding proteins ofthe present disclosure are “naked”. Put another way, the bindingproteins of the present disclosure can be un-conjugated.

In contrast, the term “conjugated” is used in the context of the presentdisclosure to refer to binding proteins of the present disclosure thatare conjugated to another compound, e.g., a toxic compound such as acytotoxic agent or radiolabel. Accordingly, in one example, the bindingproteins of the present disclosure are “conjugated”.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, 1¹³¹, 1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi, P, Pb and radioactive isotopes of Lu), chemotherapeutic agents ordrugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine,vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

Terms such as “host cell,” “host cell line,” and “host cell culture” areused interchangeably in the context of the present disclosure to referto cells into which exogenous nucleic acid has been introduced,including the progeny of such cells. Host cells include “transformants”and “transformed cells,” which include the primary transformed cell andprogeny derived therefrom without regard to the number of passages.Progeny may not be completely identical in nucleic acid content to aparent cell, but may contain mutations. Mutant progeny that have thesame function or biological activity as screened or selected for in theoriginally transformed cell are included herein.

An “isolated nucleic acid” according to the present disclosure is anucleic acid molecule that has been separated from a component of itsnatural environment. An isolated nucleic acid includes a nucleic acidmolecule contained in cells that ordinarily contain the nucleic acidmolecule, but the nucleic acid molecule is present extrachromosomally orat a chromosomal location that is different from its natural chromosomallocation.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill of those practicing in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared.

As used herein, the term “binds” in reference to the interaction of abinding protein and an antigen means that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the antigen. For example, a binding proteinrecognizes and binds to a specific antigen structure rather than toantigens generally. For example, if a binding protein binds to epitope“A”, the presence of a molecule containing epitope “A” (or free,unlabeled “A”), in a reaction containing labeled “A” and the bindingprotein, will reduce the amount of labeled “A” bound to the bindingprotein.

As used herein, the term “specifically binds” shall be taken to meanthat the binding interaction between a binding protein and DNA isdependent on detection of the DNA by the binding protein. Accordingly,the binding protein preferentially binds or recognizes DNA even whenpresent in a mixture of other molecules or organisms.

In one example, the binding protein reacts or associates morefrequently, more rapidly, with greater duration and/or with greateraffinity with DNA than it does with alternative antigens or cells. It isalso understood by reading this definition that, for example, a bindingprotein specifically binds to DNA may or may not specifically bind to asecond antigen. As such, “specific binding” does not necessarily requireexclusive binding or non-detectable binding of another antigen. The term“specifically binds” can be used interchangeably with “selectivelybinds” herein. Generally, reference herein to binding means specificbinding, and each term shall be understood to provide explicit supportfor the other term. Methods for determining specific binding will beapparent to the skilled person. For example, a binding protein of thedisclosure is contacted with DNA or an alternative antigen. Binding ofthe binding protein to DNA or alternative antigen is then determined anda binding protein that binds as set out above to the DNA rather than thealternative antigen is considered to specifically bind to DNA.

Binding proteins according to the present disclosure and compositionscomprising the same can be administered to a subject to treat variousindications. Terms such as “subject”, “patient” or “individual” areterms that can, in context, be used interchangeably in the presentdisclosure. In an example, the subject is a mammal. The mammal may be acompanion animal such as a dog or cat, or a livestock animal such as ahorse or cow. In one example, the subject is a human. For example, thesubject can be an adult. In another example, the subject can be a child.In another example, the subject can be an adolescent.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. An individual is successfully “treated”, for example, if oneor more symptoms associated with a disease are mitigated or eliminated.

As used herein, the term “prevention” includes providing prophylaxiswith respect to occurrence or recurrence of a disease in an individual.An individual may be predisposed to or at risk of developing the diseaseor disease relapse but has not yet been diagnosed with the disease orthe relapse.

The term “treatment” is used in the context of the present specificationto refer to the medical management of a patient with the intent to cure,ameliorate or stabilize a disease, pathological condition, or disorder.The term “treatment” includes active treatment, that is, treatmentdirected specifically toward the improvement of a disease, pathologicalcondition, or disorder, and also includes causal treatment, that is,treatment directed toward removal of the cause of the associateddisease, pathological condition, or disorder. In addition, the term“treatment” includes palliative treatment, that is, treatment designedfor the relief of symptoms rather than the curing of the disease,pathological condition, or disorder; prophylactic treatment, that is,treatment directed to minimizing or partially or completely inhibitingthe development of the associated disease, pathological condition, ordisorder; and supportive treatment, that is, treatment employed tosupplement another specific therapy directed toward the improvement ofthe associated disease, pathological condition, or disorder.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result. An effective amount can be provided in one or moreadministrations. In some examples of the present disclosure, the term“effective amount” is meant an amount necessary to effect treatment of adisease or condition described below. The effective amount may varyaccording to the disease or condition to be treated and also accordingto the weight, age, racial background, sex, health and/or physicalcondition and other factors relevant to the subject being treated.Typically, the effective amount will fall within a relatively broadrange (e.g. a “dosage” range) that can be determined through routinetrial and experimentation by a medical practitioner. The effectiveamount can be administered in a single dose or in a dose repeated onceor several times over a treatment period.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement of aparticular disorder (e.g. cancer). A therapeutically effective amountherein may vary according to factors such as the disease state, age,sex, and weight of the patient, and the ability of the binding proteinto elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the binding protein are outweighed by the therapeutically beneficialeffects. In the case of cancer, the therapeutically effective amount ofthe binding protein may reduce the number of cancer cells; reduce theprimary tumor size; inhibit (i.e., slow to some extent and, in someexamples, stop) cancer cell infiltration into peripheral organs; inhibit(i.e., slow to some extent and, in some examples, stop) tumormetastasis; inhibit or delay, to some extent, tumor growth or tumorprogression; and/or relieve to some extent one or more of the symptomsassociated with the cancer. To the extent the binding protein mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. For cancer therapy, efficacy in vivo can, for example,be measured by assessing the duration of survival, time to diseaseprogression (TTP), the response rates (RR), duration of response, and/orquality of life.

Deimmunized, Chimeric, Humanized, Synhumanized, Primatized and HumanAntibodies or Antigen Binding Fragments

Monoclonal antibodies are one exemplary form of binding proteincontemplated by the present disclosure. The term “monoclonal antibody”or “MAb” refers to a homogeneous antibody population capable of bindingto the same antigen(s), for example, to the same epitope within theantigen. This term is not intended to be limited as regards to thesource of the antibody or the manner in which it is made.

In an example, binding proteins encompassed by the present disclosuremay be “humanized”. A “humanized antibody” is an immunoglobulin moleculewhich contains minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, a humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions are those of a human immunoglobulin consensussequence. In an example, the humanized antibody will also comprise atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992)).

In an example, “human” binding proteins of the present disclosure caninclude amino acid residues not encoded by human sequences, e.g.mutations introduced by random or site directed mutations in vitro (inparticular mutations which involve conservative substitutions ormutations in a small number of residues of the protein, e.g. in 1, 2, 3,4 or 5 of the residues of the protein). These “human antibodies” do notnecessarily need to be generated as a result of an immune response of ahuman, rather, they can be generated using recombinant means (e.g.,screening a phage display library) and/or by a transgenic animal (e.g.,a mouse) comprising nucleic acid encoding human antibody constant and/orvariable regions and/or using guided selection (e.g., as described in orU.S. Pat. No. 5,565,332). This term also encompasses affinity maturedforms of such antibodies.

In another example, binding proteins encompassed by the presentdisclosure may be synhumanized. The term “synhumanized” refers to anantibody prepared by a method described in WO2007/019620. A synhumanizedantibody includes a variable region of an antibody, wherein the variableregion comprises FRs from a New World primate antibody variable regionand CDRs from a non-New World primate antibody variable region.

In another example, a binding protein of the present disclosure may beprimatized. A “primatized antibody” comprises variable region(s) from anantibody generated following immunization of a non-human primate (e.g.,a cynomolgus macaque). In an example, the variable regions of thenon-human primate antibody are linked to human constant regions toproduce a primatized antibody. Exemplary methods for producingprimatized antibodies are described in U.S. Pat. No. 6,113,898.

In one example, a binding protein of the disclosure is a chimericantibody or fragment. The term “chimeric antibody” or “chimeric antigenbinding fragment” refers to an antibody or fragment in which one or moreof the variable domains is from a particular species (e.g., murine, suchas mouse or rat) or belonging to a particular antibody class orsubclass, while the remainder of the antibody or fragment is fromanother species (such as, for example, human or non-human primate) orbelonging to another antibody class or subclass. In one example, achimeric antibody comprising a V_(H) and/or a V_(L) from a non-humanantibody (e.g., a murine antibody) and the remaining regions of theantibody are from a human antibody.

The present disclosure also contemplates a deimmunized antibody orantigen binding fragment thereof, e.g., as described in WO2000/34317 andWO2004/108158. De-immunized antibodies and fragments have one or moreepitopes, e.g., B cell epitopes or T cell epitopes removed (i.e.,mutated) to thereby reduce the likelihood that a subject will raise animmune response against the antibody or protein. For example, anantibody of the disclosure is analyzed to identify one or more B or Tcell epitopes and one or more amino acid residues within the epitope ismutated to thereby reduce the immunogenicity of the antibody.

Antibody Fragments Single-Domain Antibodies

In some examples, a binding protein of the disclosure is or comprises asingle-domain antibody (which is used interchangeably with the term“domain antibody” or “dAb”). A single-domain antibody is a singlepolypeptide chain comprising all or a portion of the heavy chainvariable domain of an antibody.

Single Chain Fv (scFv) Fragments

One of skill in the art will be aware that scFv's comprise V_(H) andV_(L) regions in a single polypeptide chain and a polypeptide linkerbetween the V_(H) and V_(L) which enables the scFv to form the desiredstructure for antigen binding (i.e., for the V_(H) and V_(L) of thesingle polypeptide chain to associate with one another to form a Fv).Single-chain variable fragments lack the constant Fc region found incomplete antibody molecules and therefore can have reducedimmunogenicity. Exemplary linkers comprise in excess of 12 amino acidresidues with (Gly₄Ser)₃ being one of the more favoured linkers for ascFv. Another example of a suitable linker is provided in SEQ ID NO: 31.

The present disclosure also contemplates a disulfide stabilized Fv (ordiFv or dsFv), in which a single cysteine residue is introduced into aFR of V_(H) and a FR of V_(L) and the cysteine residues linked by adisulfide bond to yield a stable Fv.

In another example, the present disclosure encompasses a dimeric scFv(di-scFV), i.e., a protein comprising two scFv molecules linked by anon-covalent or covalent linkage, e.g., by a leucine zipper domain(e.g., derived from Fos or Jun) or trimeric scFV (tri-scFv). In anotherexample, two scFv's are linked by a peptide linker of sufficient lengthto permit both scFv's to form and to bind to an antigen, e.g., asdescribed in U.S. Published Application No. 20060263367.

Diabodies, Triabodies, Tetrabodies

In some examples, an antigen binding fragment of the disclosure is orcomprises a diabody, triabody, tetrabody or higher order protein complexsuch as those described in WO98/044001 and/or WO94/007921.

For example, a diabody is a protein comprising two associatedpolypeptide chains, each polypeptide chain comprising the structureV_(L)-X-V_(H) or V_(H)-X-V_(L), wherein X is a linker comprisinginsufficient residues to permit the V_(H) and V_(L) in a singlepolypeptide chain to associate (or form an Fv) or is absent, and whereinthe V_(H) of one polypeptide chain binds to a V_(L) of the otherpolypeptide chain to form an antigen binding site, i.e., to form a Fvmolecule capable of specifically binding to one or more antigens. TheV_(L) and V_(H) can be the same in each polypeptide chain or the V_(L)and V_(H) can be different in each polypeptide chain so as to form abispecific diabody (i.e., comprising two Fv's having differentspecificity).

Other Antibodies and Antibody Fragments

Other examples of binding proteins encompassed by the present disclosureinclude:

(i) “key and hole” bispecific proteins as described in U.S. Pat. No.5,731,168;

(ii) heteroconjugate proteins, e.g., as described in U.S. Pat. No.4,676,980;

(iii) heteroconjugate proteins produced using a chemical cross-linker,e.g., as described in U.S. Pat. No. 4,676,980; and

(iv) Fab₃ (e.g., as described in EP19930302894).

Immunoglobulins and Immunoglobulin Fragments

An example of a binding protein of the present disclosure is a protein(e.g., an antibody mimetic) comprising a variable region of animmunoglobulin, such as a T cell receptor or a heavy chainimmunoglobulin (e.g., an IgNAR, a camelid antibody).

V-Like Proteins

An example of a binding protein of the disclosure is a T-cell receptor.T cell receptors have two V-domains that combine into a structuresimilar to the Fv module of an antibody. Novotny et al., Proc Natl AcadSci USA 88: 8646-8650, 1991 describes how the two V-domains of theT-cell receptor (termed alpha and beta) can be fused and expressed as asingle chain polypeptide and, further, how to alter surface residues toreduce the hydrophobicity directly analogous to an antibody scFv. Otherpublications describing production of single-chain T-cell receptors ormultimeric T cell receptors comprising two V-alpha and V-beta domainsinclude WO1999/045110 or WO2011/107595.

Other non-antibody proteins comprising antigen binding domains includeproteins with V-like domains, which are generally monomeric. Examples ofproteins comprising such V-like domains include CTLA-4, CD28 and ICOS.Further disclosure of proteins comprising such V-like domains isincluded in WO1999/045110.

Affibodies

In a further example, a binding protein of the disclosure is anaffibody. An affibody is a scaffold derived from the Z domain (antigenbinding domain) of Protein A of Staphylococcus aureus which can beengineered to bind to antigen. The Z domain consists of a three-helicalbundle of approximately 58 amino acids. Libraries have been generated byrandomization of surface residues. For further details see EP1641818.

Avimers

In a further example, a binding protein of the disclosure is an Avimer.Avimers are multidomain proteins derived from the A-domain scaffoldfamily. The native domains of approximately 35 amino acids adopt adefined disulfide bonded structure. Diversity is generated by shufflingof the natural variation exhibited by the family of A-domains. Forfurther details see WO2002/088171.

Binding Proteins

In one example, anti-DNA binding proteins according to the presentdisclosure comprise a heavy chain variable region (V_(H)) having a CDR 1as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO:3 and a CDR3 as shown in SEQ ID NO: 4. For example, an anti-DNA bindingprotein can comprise a V_(H) having a CDR1 as shown in SEQ ID NO: 1, aCDR2 as shown in SEQ ID NO: 2 and a CDR3 as shown in SEQ ID NO: 4. Inanother example, an anti-DNA binding protein can comprise a V_(H) havinga CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 3 and aCDR3 as shown in SEQ ID NO: 4.

In another example, the anti-DNA binding proteins comprise a light chainvariable region (V_(L)) having a CDR1 as shown in SEQ ID NO: 5 or SEQ IDNO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO:8. For example, an anti-DNA binding protein can comprise a V_(L) havinga CDR1 as shown in SEQ ID NO: 5, a CDR2 as shown in SEQ ID NO: 7 and aCDR3 as shown in SEQ ID NO: 8. In another example, an anti-DNA bindingprotein can comprise a V_(L) having a CDR1 as shown in SEQ ID NO: 6, aCDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8.

In another example, the anti-DNA binding proteins comprise a V_(H)having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2or SEQ ID NO: 3 and a CDR3 as shown in SEQ ID NO: 4 and a V_(L) having aCDR1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6, a CDR2 as shown in SEQ IDNO: 7 and a CDR3 as shown in SEQ ID NO: 8. For example, an anti-DNAbinding protein can comprise a V_(H) having a CDR1 as shown in SEQ IDNO: 1, a CDR2 as shown in SEQ ID NO: 2 and a CDR3 as shown in SEQ ID NO:4 and a V_(L) having a CDR1 as shown in SEQ ID NO: 5, a CDR2 as shown inSEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8. In another example, ananti-DNA binding protein can comprise a V_(H) having a CDR1 as shown inSEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2 and a CDR3 as shown in SEQID NO: 4 and a V_(L) having a CDR1 as shown in SEQ ID NO: 6, a CDR2 asshown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8. In anotherexample, an anti-DNA binding protein can comprise a V_(H) having a CDR1as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 3 and a CDR3 asshown in SEQ ID NO: 4 and a V_(L) having a CDR1 as shown in SEQ ID NO:5, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8.In another example, an anti-DNA binding protein can comprise a V_(H)having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 3and a CDR3 as shown in SEQ ID NO: 4 and a V_(L) having a CDR1 as shownin SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown inSEQ ID NO: 8.

Above exemplified binding proteins may also have CDRs assigned using theIMGT system. Accordingly, in another example, the anti-DNA bindingprotein comprises a V_(H) having a CDR1 as shown in SEQ ID NO: 9, a CDR2as shown in SEQ ID NO: 10 or SEQ ID NO: 11 and a CDR3 as shown in SEQ IDNO: 12. For example, an anti-DNA binding protein can comprise a V_(H)having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10and a CDR3 as shown in SEQ ID NO: 12. In another example, an anti-DNAbinding protein can comprise a V_(H) having a CDR1 as shown in SEQ IDNO: 9, a CDR2 as shown in SEQ ID NO: 11 and a CDR3 as shown in SEQ IDNO: 12.

In another example, the anti-DNA binding protein comprises a V_(L)having a CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, a CDR2 asshown in SEQ ID NO: 15 and a CDR3 as shown in SEQ ID NO: 16. Forexample, an anti-DNA binding protein can comprise a V_(L) having a CDR1as shown in SEQ ID NO: 13, a CDR2 as shown in SEQ ID NO: 15 and a CDR3as shown in SEQ ID NO: 16. In another example, an anti-DNA bindingprotein can comprise a V_(L) having a CDR1 as shown in SEQ ID NO: 14, aCDR2 as shown in SEQ ID NO: 15 and a CDR3 as shown in SEQ ID NO: 16.

In another example, the anti-DNA binding proteins comprises a V_(H)having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10or SEQ ID NO: 11 and a CDR3 as shown in SEQ ID NO: 12 and a V_(L) havinga CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, a CDR2 as shown inSEQ ID NO: 15 and a CDR3 as shown in SEQ ID NO: 16. For example, ananti-DNA binding protein can comprise a V_(H) having a CDR1 as shown inSEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10 and a CDR3 as shown inSEQ ID NO: 12 and a V_(L) having a CDR1 as shown in SEQ ID NO: 13, aCDR2 as shown in SEQ ID NO: 15 and a CDR3 as shown in SEQ ID NO: 16. Inanother example, an anti-DNA binding protein can comprise a V_(H) havinga CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10 and aCDR3 as shown in SEQ ID NO: 12 and a V_(L) having a CDR1 as shown in SEQID NO: 14, a CDR2 as shown in SEQ ID NO: 15 and a CDR3 as shown in SEQID NO: 16. In another example, an anti-DNA binding protein can comprisea V_(H) having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQID NO: 11 and a CDR3 as shown in SEQ ID NO: 12 and a V_(L) having a CDR1as shown in SEQ ID NO: 13, a CDR2 as shown in SEQ ID NO: 15 and a CDR3as shown in SEQ ID NO: 16. In another example, an anti-DNA bindingprotein can comprise a V_(H) having a CDR1 as shown in SEQ ID NO: 9, aCDR2 as shown in SEQ ID NO: 11 and a CDR3 as shown in SEQ ID NO: 12 anda V_(L) having a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQID NO: 15 and a CDR3 as shown in SEQ ID NO: 16.

In another example, the anti-DNA binding proteins comprise a V_(H)comprising a sequence at least 95% identical to the sequence as shown inany one of SEQ ID NOs: 17 to 23. For example, an anti-DNA bindingprotein can comprise a V_(H) comprising a sequence at least 95%identical to the sequence as shown in SEQ ID NO: 18. In another example,an anti-DNA binding protein can comprise a V_(H) comprising a sequenceat least 95% identical to the sequence as shown in SEQ ID NO: 23. Inanother example, the anti-DNA binding proteins comprise a V_(L)comprising a sequence at least 95% identical to the sequence as shown inany one of SEQ ID NOs: 24 to 29. For example, an anti-DNA bindingprotein can comprise a V_(L) comprising a sequence at least 95%identical to the sequence as shown in SEQ ID NO: 25. In another example,an anti-DNA binding protein can comprise a V_(L) comprising a sequenceat least 95% identical to the sequence as shown in SEQ ID NO: 27. Inanother example, the anti-DNA binding proteins comprise a V_(H)comprising a sequence at least 95% identical to the sequence as shown inany one of SEQ ID NOs: 17 to 23 and a V_(L) comprising a sequence atleast 95% identical to the sequence as shown in any one of SEQ ID NOs:24 to 29. For example, an anti-DNA binding protein can comprise a V_(H)comprising a sequence at least 95% identical to the sequence as shown inSEQ ID NO: 18 and a V_(L) comprising a sequence at least 95% identicalto the sequence as shown in SEQ ID NO: 25. In another example, ananti-DNA binding protein can comprise a V_(H) comprising a sequence atleast 95% identical to the sequence as shown in SEQ ID NO: 23 and aV_(L) comprising a sequence at least 95% identical to the sequence asshown in SEQ ID NO: 27. In these examples, the V_(H) and/or V_(L) can beat least 96%, at least 97%, at least 98% or at least 99% identical tothe recited SEQ ID NO.

In another example, the anti-DNA binding proteins comprise a V_(H)comprising a sequence as shown in any one of SEQ ID NOs: 17 to 23. Forexample, an anti-DNA binding protein can comprise a V_(H) comprising asequence as shown in SEQ ID NO: 18. In another example, an anti-DNAbinding protein can comprise a V_(H) comprising a sequence as shown inSEQ ID NO: 23. In another example, the anti-DNA binding proteinscomprise a V_(L) comprising a sequence as shown in any one of SEQ IDNOs: 24 to 29. For example, an anti-DNA binding protein can comprise aV_(L) comprising a sequence as shown in SEQ ID NO: 25. In anotherexample, an anti-DNA binding protein can comprise a V_(L) comprising asequence as shown in SEQ ID NO: 27. In another example, the anti-DNAbinding proteins comprise a V_(H) comprising a sequence as shown in anyone of SEQ ID NOs: 17 to 23 and a V_(L) comprising a sequence as shownin any one of SEQ ID NOs: 24 to 29. For example, an anti-DNA bindingprotein can comprise a V_(H) comprising a sequence as shown in SEQ IDNO: 18 and a V_(L) comprising a sequence as shown in SEQ ID NO: 25. Inanother example, an anti-DNA binding protein can comprise a V_(H)comprising a sequence as shown in SEQ ID NO: 23 and a V_(L) comprising asequence as shown in SEQ ID NO: 27.

In an example, the anti-DNA binding protein can be a cell penetratinganti-DNA Fv fragment having an antigen binding domain, wherein theantigen binding domain binds to or specifically binds to DNA. Forexample, the Fv can bind the same epitope as a binding protein having aV_(H) comprising an amino acid sequence as shown in SEQ ID NO: 48 and aV_(L) comprising an amino acid sequence as shown in SEQ ID NO: 49. Inanother example, the Fv can bind the same epitope as a di-scFv having anamino acid sequence as shown in SEQ ID NO: 50. In an example, the Fvcomprises a V_(H) having a CDR1 as shown in SEQ ID NO: 1, a CDR2 asshown in SEQ ID NO: 2 or SEQ ID NO: 3 and a CDR3 as shown in SEQ ID NO:4. For example, an Fv can comprise a V_(H) having a CDR1 as shown in SEQID NO: 1, a CDR2 as shown in SEQ ID NO: 2 and a CDR3 as shown in SEQ IDNO: 4. In another example, an Fv can comprise a V_(H) having a CDR1 asshown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 3 and a CDR3 asshown in SEQ ID NO: 4.

In another example, the Fv comprises a V_(L) having a CDR1 as shown inSEQ ID NO: 5 or SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3as shown in SEQ ID NO: 8. For example, an Fv can comprise a V_(L) havinga CDR1 as shown in SEQ ID NO: 5, a CDR2 as shown in SEQ ID NO: 7 and aCDR3 as shown in SEQ ID NO: 8. In another example, an anti-DNA bindingprotein can comprise a V_(L) having a CDR1 as shown in SEQ ID NO: 6, aCDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8.

In another example, the Fv comprises a V_(H) having a CDR1 as shown inSEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3 and a CDR3as shown in SEQ ID NO: 4 and a V_(L) having a CDR1 as shown in SEQ IDNO: 5 or SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 asshown in SEQ ID NO: 8. For example, an Fv can comprise a V_(H) having aCDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2 and aCDR3 as shown in SEQ ID NO: 4 and a V_(L) having a CDR1 as shown in SEQID NO: 5, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ IDNO: 8. In another example, an Fv can comprise a V_(H) having a CDR1 asshown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2 and a CDR3 asshown in SEQ ID NO: 4 and a V_(L) having a CDR1 as shown in SEQ ID NO:6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8.In another example, an Fv can comprise a V_(H) having a CDR1 as shown inSEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 3 and a CDR3 as shown in SEQID NO: 4 and a V_(L) having a CDR1 as shown in SEQ ID NO: 5, a CDR2 asshown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8. In anotherexample, an Fv can comprise a V_(H) having a CDR1 as shown in SEQ ID NO:1, a CDR2 as shown in SEQ ID NO: 3 and a CDR3 as shown in SEQ ID NO: 4and a V_(L) having a CDR1 as shown in SEQ ID NO: 6, a CDR2 as shown inSEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8.

Above exemplified Fv may also have CDRs assigned using the IMGT system.Accordingly, in another example, the Fv comprises a V_(H) having a CDR1as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO:11 and a CDR3 as shown in SEQ ID NO: 12. For example, an Fv can comprisea V_(H) having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQID NO: 10 and a CDR3 as shown in SEQ ID NO: 12. In another example, anFv can comprise a V_(H) having a CDR1 as shown in SEQ ID NO: 9, a CDR2as shown in SEQ ID NO: 11 and a CDR3 as shown in SEQ ID NO: 12.

In another example, the Fv comprises a V_(L) having a CDR1 as shown inSEQ ID NO: 13 or SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 15 and aCDR3 as shown in SEQ ID NO: 16. For example, an Fv can comprise a V_(L)having a CDR1 as shown in SEQ ID NO: 13, a CDR2 as shown in SEQ ID NO:15 and a CDR3 as shown in SEQ ID NO: 16. In another example, an Fv cancomprise a V_(L) having a CDR1 as shown in SEQ ID NO: 14, a CDR2 asshown in SEQ ID NO: 15 and a CDR3 as shown in SEQ ID NO: 16.

In another example, the Fv comprises a V_(H) having a CDR1 as shown inSEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11 and aCDR3 as shown in SEQ ID NO: 12 and a V_(L) having a CDR1 as shown in SEQID NO: 13 or SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 15 and a CDR3as shown in SEQ ID NO: 16. For example, an Fv can comprise a V_(H)having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10and a CDR3 as shown in SEQ ID NO: 12 and a V_(L) having a CDR1 as shownin SEQ ID NO: 13, a CDR2 as shown in SEQ ID NO: 15 and a CDR3 as shownin SEQ ID NO: 16. In another example, an Fv can comprise a V_(H) havinga CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10 and aCDR3 as shown in SEQ ID NO: 12 and a V_(L) having a CDR1 as shown in SEQID NO: 14, a CDR2 as shown in SEQ ID NO: 15 and a CDR3 as shown in SEQID NO: 16. In another example, an Fv can comprise a V_(H) having a CDR1as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11 and a CDR3 asshown in SEQ ID NO: 12 and a V_(L) having a CDR1 as shown in SEQ ID NO:13, a CDR2 as shown in SEQ ID NO: 15 and a CDR3 as shown in SEQ ID NO:16. In another example, an Fv can comprise a V_(H) having a CDR1 asshown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11 and a CDR3 asshown in SEQ ID NO: 12 and a V_(L) having a CDR1 as shown in SEQ ID NO:14, a CDR2 as shown in SEQ ID NO: 15 and a CDR3 as shown in SEQ ID NO:16.

In another example, the Fv comprises a V_(H) comprising a sequence atleast 95% identical to the sequence as shown in any one of SEQ ID NOs:17 to 23. For example, an Fv can comprise a V_(H) comprising a sequenceat least 95% identical to the sequence as shown in SEQ ID NO: 18. Inanother example, an Fv can comprise a V_(H) comprising a sequence atleast 95% identical to the sequence as shown in SEQ ID NO: 21. Inanother example, an Fv can comprise a V_(H) comprising a sequence atleast 95% identical to the sequence as shown in SEQ ID NO: 23. Inanother example, the Fv comprises a V_(L) comprising a sequence at least95% identical to the sequence as shown in any one of SEQ ID NOs: 24 to29. For example, an Fv can comprise a V_(L) comprising a sequence atleast 95% identical to the sequence as shown in SEQ ID NO: 25. Inanother example, an Fv can comprise a V_(L) comprising a sequence atleast 95% identical to the sequence as shown in SEQ ID NO: 27. Inanother example, the Fv comprises a V_(H) comprising a sequence at least95% identical to the sequence as shown in any one of SEQ ID NOs: 17 to23 and a V_(L) comprising a sequence at least 95% identical to thesequence as shown in any one of SEQ ID NOs: 24 to 29. For example, an Fvcan comprise a V_(H) comprising a sequence at least 95% identical to thesequence as shown in SEQ ID NO: 18 and a V_(L) comprising a sequence atleast 95% identical to the sequence as shown in SEQ ID NO: 25. Inanother example, an Fv can comprise a V_(H) comprising a sequence atleast 95% identical to the sequence as shown in SEQ ID NO: 21 and aV_(L) comprising a sequence at least 95% identical to the sequence asshown in SEQ ID NO: 27. In another example, an Fv can comprise a V_(H)comprising a sequence at least 95% identical to the sequence as shown inSEQ ID NO: 23 and a V_(L) comprising a sequence at least 95% identicalto the sequence as shown in SEQ ID NO: 27. In these examples, the V_(H)and/or V_(L) can be at least 96%, at least 97%, at least 98% or at least99% identical to the recited SEQ ID NO. In these examples, the Fv canhave an above referenced combination of CDRs. For example, an Fv cancomprise a V_(H) comprising a sequence at least 95% identical to thesequence as shown in SEQ ID NO: 21 and a V_(L) comprising a sequence atleast 95% identical to the sequence as shown in SEQ ID NO: 27, whereinthe V_(H) has a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ IDNO: 3 and a CDR3 as shown in SEQ ID NO: 4 and the V_(L) has a CDR1 asshown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 asshown in SEQ ID NO: 8.

In another example, the Fv comprises a V_(H) comprising a sequence asshown in any one of SEQ ID NOs: 17 to 23. For example, an Fv cancomprise a V_(H) comprising a sequence as shown in SEQ ID NO: 18. Inanother example, an Fv can comprise a V_(H) comprising a sequence asshown in SEQ ID NO: 21. In another example, an Fv can comprise a V_(H)comprising a sequence as shown in SEQ ID NO: 23. In another example, theFv comprises a V_(L) comprising a sequence as shown in any one of SEQ IDNOs: 24 to 29. For example, an Fv can comprise a V_(L) comprising asequence as shown in SEQ ID NO: 25. In another example, an Fv cancomprise a V_(L) comprising a sequence as shown in SEQ ID NO: 27. Inanother example, the Fv comprises a V_(H) comprising a sequence as shownin any one of SEQ ID NOs: 17 to 23 and a V_(L) comprising a sequence asshown in any one of SEQ ID NOs: 24 to 29. For example, an Fv cancomprise a V_(H) comprising a sequence as shown in SEQ ID NO: 18 and aV_(L) comprising a sequence as shown in SEQ ID NO: 25. In anotherexample, an Fv can comprise a V_(H) comprising a sequence as shown inSEQ ID NO: 21 and a V_(L) comprising a sequence as shown in SEQ ID NO:27. In another example, an Fv can comprise a V_(H) comprising a sequenceas shown in SEQ ID NO: 23 and a V_(L) comprising a sequence as shown inSEQ ID NO: 27.

In another example, the Fv has improved manufacturability compared to abinding protein having a V_(H) comprising an amino acid sequence asshown in SEQ ID NO: 48 and a V_(L) comprising an amino acid sequence asshown in SEQ ID NO: 49. In another example, the Fv has improvedmanufacturability compared to a di-scFv having an amino acid sequence asshown in SEQ ID NO: 50.

Improved manufacturability encompasses post translational modificationsor increased chemical stability relating to reduced numbers ofdeamidation sites, aspartate isomerization sites, oxidation sites suchas methionine and tryptophan, free-cysteine thiol groups, N &O-glycosylation sites, the presence of C-terminal lysine and/orisoelectric point.

In an example, the Fv comprises less asparagine in the V_(H) and/orV_(L) compared with a binding protein having a V_(H) comprising an aminoacid sequence as shown in SEQ ID NO: 48 and a V_(L) comprising an aminoacid sequence as shown in SEQ ID NO: 49. In another example, the Fvcomprises less asparagine in the V_(H) and/or V_(L) compared with adi-scFv having an amino acid sequence as shown in SEQ ID NO: 50.

In an example, the Fv comprises less methionine in the V_(H) and/orV_(L) compared with a binding protein having a V_(H) comprising an aminoacid sequence as shown in SEQ ID NO: 48 and a V_(L) comprising an aminoacid sequence as shown in SEQ ID NO: 49. In another example, the Fvcomprises less methionine in the V_(H) and/or V_(L) compared with adi-scFv having an amino acid sequence as shown in SEQ ID NO: 50.

In an example, the Fv comprises less tryptophan in the V_(H) and/orV_(L) compared with a binding protein having a V_(H) comprising an aminoacid sequence as shown in SEQ ID NO: 48 and a V_(L) comprising an aminoacid sequence as shown in SEQ ID NO: 49. In another example, the Fvcomprises less tryptophan in the V_(H) and/or V_(L) compared with adi-scFv having an amino acid sequence as shown in SEQ ID NO: 50.

In an example, the Fv comprises less aspartic acid in the V_(H) and/orV_(L) compared with a binding protein having a V_(H) comprising an aminoacid sequence as shown in SEQ ID NO: 48 and a V_(L) comprising an aminoacid sequence as shown in SEQ ID NO: 49. In another example, the Fvcomprises less aspartic acid in the V_(H) and/or V_(L) compared with adi-scFv having an amino acid sequence as shown in SEQ ID NO: 50.

In an example, the physical stability of the Fv is greater than abinding protein having a V_(H) comprising an amino acid sequence asshown in SEQ ID NO: 48 and a V_(L) comprising an amino acid sequence asshown in SEQ ID NO: 49. In another example, the physical stability ofthe Fv is greater than a di-scFv having an amino acid sequence as shownin SEQ ID NO: 50.

Physical stability can include propensity for aggregation in solution.The term “aggregation” is used in the context of the present disclosureto refer to protein self-association, which can occur in multipleenvironments, from cell culture and fermentation, to isolation,purification and formulation processes. For example, the term“aggregation” can be used when describing the formation of inclusions;the accumulation of protein in “insoluble” fractions following cellfractionation; the appearance of turbidity, protein precipitation orformation of particles in samples; or the formation of small solubleoligomers amongst others.

Accordingly, in the above referenced examples, the physical stability ofa Fv can be based on its physical stability in solution, whereinprecipitation of the Fv from solution indicates that the Fv has becomeunstable. To assess physical stability, solutions comprising a Fvaccording to the present disclosure or either a binding protein having aV_(H) comprising an amino acid sequence as shown in SEQ ID NO: 48 and aV_(L) comprising an amino acid sequence as shown in SEQ ID NO: 49 or adi-scFv comprising an amino acid sequence as shown in SEQ ID NO: 50 canbe incubated at 4° C. and assessed visually for precipitation at twoweeks, four weeks, 12 weeks, six months and 12 months.

In an example, the physical stability of an Fv according to the presentdisclosure is greater than a binding protein having a V_(H) comprisingan amino acid sequence as shown in SEQ ID NO: 48 and a V_(L) comprisingan amino acid sequence as shown in SEQ ID NO: 49 or a di-scFv comprisingan amino acid sequence as shown in SEQ ID NO: 50 when the Fv remains insolution at 4° C. for at least four weeks. In an example, the physicalstability of an Fv according to the present disclosure is greater than abinding protein having a V_(H) comprising an amino acid sequence asshown in SEQ ID NO: 48 and a V_(L) comprising an amino acid sequence asshown in SEQ ID NO: 49 or a di-scFv comprising an amino acid sequence asshown in SEQ ID NO: 50 when the Fv remains in solution at 4° C. for atleast six months.

In another example, the Fv has reduced immunogenicity in a human subjectcompared to a binding protein having a V_(H) comprising an amino acidsequence as shown in SEQ ID NO: 48 and a V_(L) comprising an amino acidsequence as shown in SEQ ID NO: 49. For example, an Fv can have reducedimmunogenicity compared to a binding protein having a V_(H) comprisingan amino acid sequence as shown in SEQ ID NO: 48 and a V_(L) comprisingan amino acid sequence as shown in SEQ ID NO: 49 when immunogenicity ismeasure via enzyme-linked immunosorbent assay (ELISA). In anotherexample, an Fv can have reduced immunogenicity compared to a bindingprotein having a V_(H) comprising an amino acid sequence as shown in SEQID NO: 48 and a V_(L) comprising an amino acid sequence as shown in SEQID NO: 49 when immunogenicity is measure via Surface Plasmon Resonance.

In another example, the capacity of the Fv to penetrate cells is greaterthan a binding protein having a V_(H) comprising an amino acid sequenceas shown in SEQ ID NO: 48 and a V_(L) comprising an amino acid sequenceas shown in SEQ ID NO: 49. In another example, the capacity of the Fv topenetrate cells is greater than a di-scFv having an amino acid sequenceas shown in SEQ ID NO: 50. In another example, the capacity of the Fv topenetrate cell nuclei is greater than a binding protein having a V_(H)comprising an amino acid sequence as shown in SEQ ID NO: 48 and a V_(L)comprising an amino acid sequence as shown in SEQ ID NO: 49. In anotherexample, the capacity of the Fv to penetrate cell nuclei is greater thana di-scFv having an amino acid sequence as shown in SEQ ID NO: 50. Forexample, the di-scFv can comprise an amino acid sequence as shown in SEQID NO: 36. In another example, the di-scFv can comprise an amino acidsequence as shown in SEQ ID NO: 41. In another example, the di-scFv cancomprise an amino acid sequence as shown in SEQ ID NO: 43. In the abovereferenced examples, the capacity of a binding protein to penetratecells or cell nuclei can be measured using a colorimetric assay. Forexample, cells can be treated with control media, a binding proteinaccording to the present disclosure or either a binding protein having aV_(H) comprising an amino acid sequence as shown in SEQ ID NO: 48 and aV_(L) comprising an amino acid sequence as shown in SEQ ID NO: 49 or adi-scFv having an amino acid sequence as shown in SEQ ID NO: 50 for onehour. Cells are then washed, fixed, blocked with 1% BSA-TBST, and thenprobed with protein L for one hour. Cells are then washed and incubatedwith an anti-protein L primary antibody for one hour. After anotherround of washing, cells are incubated with an alkalinephosphatase-conjugated secondary antibody for one hour. Finally, cellsare washed and signal is developed by addition of NBT/BCIP. Signaldevelopment is stopped by removal of NBT/BCIP and washing once distinctnuclear stain is identifiable in any of the samples. Nuclear and orcellular staining is then measured using Image J.

In an example, an Fv providing nuclear staining having reciprocalintensity of at least 190 absorbance units (au) has greater capacity topenetrate cell nuclei. In an example, an Fv providing nuclear staininghaving reciprocal intensity of at least 200 au has greater capacity topenetrate cell nuclei. In an example, an Fv providing nuclear staininghaving reciprocal intensity of at least 210 au has greater capacity topenetrate cell nuclei. In an example, an Fv providing nuclear staininghaving reciprocal intensity of at least 220 au has greater capacity topenetrate cell nuclei. In another example, the capacity of an Fv topenetrate cell nuclei can be assessed by measuring florescence inindividual cells. In an example, an Fv providing nuclear staining havingreciprocal intensity of at least 190 au in at least 20 cells has greatercapacity to penetrate cell nuclei. In another example, an Fv providingnuclear staining having reciprocal intensity of at least 190 au in atleast 30 cells has greater capacity to penetrate cell nuclei. In anotherexample, an Fv providing nuclear staining having reciprocal intensity ofat least 190 au in at least 40 cells has greater capacity to penetratecell nuclei. In another example, an Fv providing nuclear staining havingreciprocal intensity of at least 200 au in at least 20 cells has greatercapacity to penetrate cell nuclei. In another example, an Fv providingnuclear staining having reciprocal intensity of at least 200 au in atleast 30 cells has greater capacity to penetrate cell nuclei. In anotherexample, an Fv providing nuclear staining having reciprocal intensity ofat least 200 au in at least 50 cells has greater capacity to penetratecell nuclei. In another example, an Fv providing nuclear staining havingreciprocal intensity of at least 200 au in at least 70 cells has greatercapacity to penetrate cell nuclei. In another example, an Fv providingnuclear staining having reciprocal intensity of at least 200 au in atleast 80 cells has greater capacity to penetrate cell nuclei.

In another example, the Fv has higher specificity for DNA than a bindingprotein having a V_(H) comprising an amino acid sequence as shown in SEQID NO: 48 and a V_(L) comprising an amino acid sequence as shown in SEQID NO: 49. In another example, the Fv has higher specificity for DNAthan a di-scFv having an amino acid sequence as shown in SEQ ID NO: 50.

In another example, the Fv has lower cross-reactivity (i.e. the abilityof an Fv to react with similar antigenic sites on different proteins)compared to a binding protein having a V_(H) comprising an amino acidsequence as shown in SEQ ID NO: 48 and a V_(L) comprising an amino acidsequence as shown in SEQ ID NO: 49. In another example, the Fv has lowercross-reactivity with other targets compared to a di-scFv having anamino acid sequence as shown in SEQ ID NO: 50. In this example,cross-reactivity of an Fv can be measured using various methods. In anexample, cross-reactivity is assessed via ELISA.

In another example, the Fv has higher binding affinity for DNA than abinding protein having a V_(H) comprising an amino acid sequence asshown in SEQ ID NO: 48 and a V_(L) comprising an amino acid sequence asshown in SEQ ID NO: 49. In another example, the Fv has higher bindingaffinity for DNA than a di-scFv having an amino acid sequence as shownin SEQ ID NO: 50.

In the above referenced examples, the affinity of an Fv for DNA can bemeasured using various methods. In an example, the dissociation constant(K_(D)) or association constant (K_(A)) or equilibrium constant (K_(D))of a binding protein for DNA is determined. These constants for abinding protein are, in one example, measured by a radiolabeled orfluorescently-labelled DNA-binding assay. This assay equilibrates thebinding protein with a minimal concentration of labelled DNA (or asoluble form thereof, e.g., comprising an extracellular region of DNAfused to an Fc region) in the presence of a titration series ofunlabelled DNA. Following washing to remove unbound DNA, the amount oflabel is determined.

Affinity measurements can be determined by standard methodology forantibody reactions, for example, immunoassays, surface plasmon resonance(SPR) (Rich and Myszka Curr. Opin. Biotechnol 11:54, 2000; EnglebienneAnalyst. 123: 1599, 1998), isothermal titration calorimetry (ITC) orother kinetic interaction assays known in the art.

In one example, the constants are measured by using surface plasmonresonance assays, e.g., using BIAcore surface plasmon resonance(BIAcore, Inc., Piscataway, N.J.) with immobilized DNA. Exemplary SPRmethods are described in U.S. Pat. No. 7,229,619.

In some embodiments, the binding affinity for DNA of the Fv is betweenabout 5 nM and about 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, or 1 fM.

In an example, Fv encompassed by the present disclosure have a bindingaffinity for DNA comparable to about 5 nM or less, or about 4.9 nM, orabout 4.8 nM, or about 4.7 nM, or about 4.6 nM, or about 4.7 nM, orabout 4.6 nM, or about 4.5 nM, or about 4.4 nM, or about 4.3 nM, orabout 4.2 nM, or about 4.1 nM, or about 4.0 nM, or about 3.9 nM, orabout 3.8 nM, or about 3.7 nM, or about 3.6 nM, or about 3.5 nM, orabout 3.4 nM, or about 3.3 nM, or about 3.2 nM, or about 3.1 nM, orabout 3.0 nM.

In other examples, subject Fv can have a binding affinity for DNAcomparable to about 100 pM, or about 150 pM, or about 200 pM, or about250 pM, or about 300 pM, or about 350 pM, or about 400 pM, or about 450pM, or about 466 pM as measured by surface plasmon resonance (e.g. usinga BIAcore 3000 instrument).

In the other examples, the affinity of a binding protein for DNA can bemeasured using Isothermal Titration Microcalorimetry.

In an example, the Fv comprises a linker. Various suitable linkers andmethods for their design have been described previously (e.g. U.S. Pat.No. 4,946,778; WO 1994/012520; and U.S. Pat. No. 4,704,692). In anexample, the Fv comprises a glycine-serine (GS) linker. For example, theGS linker can comprise (GGGGS)₃ (SEQ ID NO: 67). In an example, the Fvcomprises a linker having the sequence shown in SEQ ID NO: 30. Inanother example, the Fv comprises a linker having the sequence shown inSEQ ID NO: 31. In another example, the Fv comprises linkers having thesequences shown in SEQ ID NO: 30 and SEQ ID NO: 31.

In an example, the V_(H) and V_(L) of the Fv can be in a singlepolypeptide chain. In another example, the Fv lacks an Fc region. Forexample, the Fv can be a single chain Fv fragment (scFv), a dimeric scFv(di-scFv), a trimeric scFv (tri-scFv). In an example, the Fv is an scFv.In another example, the Fv is a di-scFv. In another example, the Fv is atri-scFv.

In another example, the scFv, di-scFv or tri-scFv can be linked to aconstant region of an antibody, Fc or a heavy chain constant domainC_(H)2 and/or C_(H)3.

In an example, the present disclosure encompasses a cell penetratingdi-scFv having an antigen binding domain, wherein the antigen bindingdomain binds to or specifically binds to DNA.

In an example, a di-scFv according to the present disclosure comprisesan amino acid sequence at least 95% identical to the sequence as shownin any one of SEQ ID NOs: 32 to 47. In an example, the di-scFv comprisesan amino acid sequence at least 95% identical to the sequence shown inSEQ ID NO: 32. In an example, the di-scFv comprises an amino acidsequence at least 95% identical to the sequence shown in SEQ ID NO: 33.In an example, the di-scFv comprises an amino acid sequence at least 95%identical to the sequence shown in SEQ ID NO: 34. In an example, thedi-scFv comprises an amino acid sequence at least 95% identical to thesequence shown in SEQ ID NO: 35. In an example, the di-scFv comprises anamino acid sequence at least 95% identical to the sequence shown in SEQID NO: 36. In an example, the di-scFv comprises an amino acid sequenceat least 95% identical to the sequence shown in SEQ ID NO: 37. In anexample, the di-scFv comprises an amino acid sequence at least 95%identical to the sequence shown in SEQ ID NO: 38. In an example, thedi-scFv comprises an amino acid sequence at least 95% identical to thesequence shown in SEQ ID NO: 39. In an example, the di-scFv comprises anamino acid sequence at least 95% identical to the sequence shown in SEQID NO: 40. In an example, the di-scFv comprises an amino acid sequenceat least 95% identical to the sequence shown in SEQ ID NO: 41. In anexample, the di-scFv comprises an amino acid sequence at least 95%identical to the sequence shown in SEQ ID NO: 42. In an example, thedi-scFv comprises an amino acid sequence at least 95% identical to thesequence shown in SEQ ID NO: 43. In an example, the di-scFv comprises anamino acid sequence at least 95% identical to the sequence shown in SEQID NO: 44. In an example, the di-scFv comprises an amino acid sequenceat least 95% identical to the sequence shown in SEQ ID NO: 45. In anexample, the di-scFv comprises an amino acid sequence at least 95%identical to the sequence shown in SEQ ID NO: 46. In an example, thedi-scFv comprises an amino acid sequence at least 95% identical to thesequence shown in SEQ ID NO: 47. For example, the di-scFv comprises anamino acid sequence at least 95% identical to the amino acid sequenceshown in any one of SEQ ID NOs: 32, 36, 41 or 43. In these examples,amino acid sequences can be at least 96%, at least 97%, at least 98% orat least 99% identical to the recited SEQ ID NO.

In an example, a di-scFv according to the present disclosure comprisesan amino acid sequence as shown in any one of SEQ ID NOs: 32 to 47. Inan example, the di-scFv comprises an amino acid sequence as shown in SEQID NO: 32. In an example, the di-scFv comprises an amino acid sequenceas shown in SEQ ID NO: 33. In an example, the di-scFv comprises an aminoacid sequence as shown in SEQ ID NO: 34. In an example, the di-scFvcomprises an amino acid sequence as shown in SEQ ID NO: 35. In anexample, the di-scFv comprises an amino acid sequence as shown in SEQ IDNO: 36. In an example, the di-scFv comprises an amino acid sequence asshown in SEQ ID NO: 37. In an example, the di-scFv comprises an aminoacid sequence as shown in SEQ ID NO: 38. In an example, the di-scFvcomprises an amino acid sequence as shown in SEQ ID NO: 39. In anexample, the di-scFv comprises an amino acid sequence as shown in SEQ IDNO: 40. In an example, the di-scFv comprises an amino acid sequence asshown in SEQ ID NO: 41. In an example, the di-scFv comprises an aminoacid sequence as shown in SEQ ID NO: 42. In an example, the di-scFvcomprises an amino acid sequence as shown in SEQ ID NO: 43. In anexample, the di-scFv comprises an amino acid sequence as shown in SEQ IDNO: 44. In an example, the di-scFv comprises an amino acid sequence asshown in SEQ ID NO: 45. In an example, the di-scFv comprises an aminoacid sequence as shown in SEQ ID NO: 46. In an example, the di-scFvcomprises an amino acid sequence as shown in SEQ ID NO: 47. For example,the di-scFv can comprise an amino acid sequence as shown in any one ofSEQ ID NOs: 32, 36, 41 and 43.

In another example, the V_(H) and V_(L) of the binding protein are in aseparate polypeptide chain. For example, the binding protein can be adiabody, triabody, tetrabody, Fab, F(ab′)₂. In another example, thebinding protein can be an Fv which comprises a V_(H) and V_(L) inseparate polypeptide chains. In these examples, the binding proteins maybe linked to a constant region of an antibody, Fc or a heavy chainconstant domain C_(H)2 and/or C_(H)3. In another example, the bindingprotein can be an intact antibody. Accordingly, in an example, thepresent disclosure encompasses an antibody having an antigen bindingdomain, wherein the antigen binding domain binds to or specificallybinds to DNA. For example, the antibody can bind the same epitope as abinding protein having a V_(H) comprising an amino acid sequence asshown in SEQ ID NO: 48 and a V_(L) comprising an amino acid sequence asshown in SEQ ID NO: 49. In another example, the antibody can bind thesame epitope as a di-scFv having an amino acid sequence as shown in SEQID NO: 50. In an example, the antibody comprises a V_(H) having a CDR1as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO:3 and a CDR3 as shown in SEQ ID NO: 4. For example, an antibody cancomprise a V_(H) having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shownin SEQ ID NO: 2 and a CDR3 as shown in SEQ ID NO: 4. In another example,an antibody can comprise a V_(H) having a CDR1 as shown in SEQ ID NO: 1,a CDR2 as shown in SEQ ID NO: 3 and a CDR3 as shown in SEQ ID NO: 4.

In another example, the antibody comprises a V_(L) having a CDR1 asshown in SEQ ID NO: 5 or SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7and a CDR3 as shown in SEQ ID NO: 8. For example, an antibody cancomprise a V_(L) having a CDR1 as shown in SEQ ID NO: 5, a CDR2 as shownin SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO: 8. In another example,an anti-DNA binding protein can comprise a V_(L) having a CDR1 as shownin SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown inSEQ ID NO: 8.

In another example, the antibody comprises a V_(H) having a CDR1 asshown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3and a CDR3 as shown in SEQ ID NO: 4 and a V_(L) having a CDR1 as shownin SEQ ID NO: 5 or SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and aCDR3 as shown in SEQ ID NO: 8. For example, an antibody can comprise aV_(H) having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ IDNO: 2 and a CDR3 as shown in SEQ ID NO: 4 and a V_(L) having a CDR1 asshown in SEQ ID NO: 5, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 asshown in SEQ ID NO: 8. In another example, an antibody can comprise aV_(H) having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ IDNO: 2 and a CDR3 as shown in SEQ ID NO: 4 and a V_(L) having a CDR1 asshown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 asshown in SEQ ID NO: 8. In another example, an antibody can comprise aV_(H) having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ IDNO: 3 and a CDR3 as shown in SEQ ID NO: 4 and a V_(L) having a CDR1 asshown in SEQ ID NO: 5, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 asshown in SEQ ID NO: 8. In another example, an antibody can comprise aV_(H) having a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ IDNO: 3 and a CDR3 as shown in SEQ ID NO: 4 and a V_(L) having a CDR1 asshown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 asshown in SEQ ID NO: 8.

Above exemplified antibodies may also have CDRs assigned using the IMGTsystem. Accordingly, in another example, the antibody comprises a V_(H)having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10or SEQ ID NO: 11 and a CDR3 as shown in SEQ ID NO: 12. For example, anantibody can comprise a V_(H) having a CDR1 as shown in SEQ ID NO: 9, aCDR2 as shown in SEQ ID NO: 10 and a CDR3 as shown in SEQ ID NO: 12. Inanother example, an antibody can comprise a V_(H) having a CDR1 as shownin SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 11 and a CDR3 as shown inSEQ ID NO: 12.

In another example, the antibody comprises a V_(L) having a CDR1 asshown in SEQ ID NO: 13 or SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO:15 and a CDR3 as shown in SEQ ID NO: 16. For example, an antibody cancomprise a V_(L) having a CDR1 as shown in SEQ ID NO: 13, a CDR2 asshown in SEQ ID NO: 15 and a CDR3 as shown in SEQ ID NO: 16. In anotherexample, an antibody can comprise a V_(L) having a CDR1 as shown in SEQID NO: 14, a CDR2 as shown in SEQ ID NO: 15 and a CDR3 as shown in SEQID NO: 16.

In another example, the antibody comprises a V_(H) having a CDR1 asshown in SEQ ID NO: 9, a CDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11and a CDR3 as shown in SEQ ID NO: 12 and a V_(L) having a CDR1 as shownin SEQ ID NO: 13 or SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 15 anda CDR3 as shown in SEQ ID NO: 16. For example, an antibody can comprisea V_(H) having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQID NO: 10 and a CDR3 as shown in SEQ ID NO: 12 and a V_(L) having a CDR1as shown in SEQ ID NO: 13, a CDR2 as shown in SEQ ID NO: 15 and a CDR3as shown in SEQ ID NO: 16. In another example, an antibody can comprisea V_(H) having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQID NO: 10 and a CDR3 as shown in SEQ ID NO: 12 and a V_(L) having a CDR1as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 15 and a CDR3as shown in SEQ ID NO: 16. In another example, an antibody can comprisea V_(H) having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQID NO: 11 and a CDR3 as shown in SEQ ID NO: 12 and a V_(L) having a CDR1as shown in SEQ ID NO: 13, a CDR2 as shown in SEQ ID NO: 15 and a CDR3as shown in SEQ ID NO: 16. In another example, an antibody can comprisea V_(H) having a CDR1 as shown in SEQ ID NO: 9, a CDR2 as shown in SEQID NO: 11 and a CDR3 as shown in SEQ ID NO: 12 and a V_(L) having a CDR1as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 15 and a CDR3as shown in SEQ ID NO: 16.

In another example, the antibody comprises a V_(H) comprising a sequenceat least 95% identical to the sequence as shown in any one of SEQ IDNOs: 17 to 23. For example, an antibody can comprise a V_(H) comprisinga sequence at least 95% identical to the sequence as shown in SEQ ID NO:18. In another example, an antibody can comprise a V_(H) comprising asequence at least 95% identical to the sequence as shown in SEQ ID NO:21. In another example, an antibody can comprise a V_(H) comprising asequence at least 95% identical to the sequence as shown in SEQ ID NO:23. In another example, the antibody comprises a V_(L) comprising asequence at least 95% identical to the sequence as shown in any one ofSEQ ID NOs: 24 to 29. For example, an antibody can comprise a V_(L)comprising a sequence at least 95% identical to the sequence as shown inSEQ ID NO: 25. In another example, an antibody can comprise a V_(L)comprising a sequence at least 95% identical to the sequence as shown inSEQ ID NO: 27. In another example, the antibody comprises a V_(H)comprising a sequence at least 95% identical to the sequence as shown inany one of SEQ ID NOs: 17 to 23 and a V_(L) comprising a sequence atleast 95% identical to the sequence as shown in any one of SEQ ID NOs:24 to 29. For example, an antibody can comprise a V_(H) comprising asequence at least 95% identical to the sequence as shown in SEQ ID NO:18 and a V_(L) comprising a sequence at least 95% identical to thesequence as shown in SEQ ID NO: 25. In another example, an antibody cancomprise a V_(H) comprising a sequence at least 95% identical to thesequence as shown in SEQ ID NO: 21 and a V_(L) comprising a sequence atleast 95% identical to the sequence as shown in SEQ ID NO: 27. Inanother example, an antibody can comprise a V_(H) comprising a sequenceat least 95% identical to the sequence as shown in SEQ ID NO: 23 and aV_(L) comprising a sequence at least 95% identical to the sequence asshown in SEQ ID NO: 27. In these examples, the V_(H) and/or V_(L) can beat least 96%, at least 97%, at least 98% or at least 99% identical tothe recited SEQ ID NO. In these examples, the antibody can have an abovereferenced combination of CDRs. For example, an antibody can comprise aV_(H) comprising a sequence at least 95% identical to the sequence asshown in SEQ ID NO: 21 and a V_(L) comprising a sequence at least 95%identical to the sequence as shown in SEQ ID NO: 27, wherein the V_(H)has a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 3 anda CDR3 as shown in SEQ ID NO: 4 and the V_(L) has a CDR1 as shown in SEQID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ IDNO: 8.

In another example, the antibody comprises a V_(H) comprising a sequenceas shown in any one of SEQ ID NOs: 17 to 23. For example, an antibodycan comprise a V_(H) comprising a sequence as shown in SEQ ID NO: 18. Inanother example, an antibody can comprise a V_(H) comprising a sequenceas shown in SEQ ID NO: 21. In another example, an antibody can comprisea V_(H) comprising a sequence as shown in SEQ ID NO: 23. In anotherexample, the antibody comprises a V_(L) comprising a sequence as shownin any one of SEQ ID NOs: 24 to 29. For example, an antibody cancomprise a V_(L) comprising a sequence as shown in SEQ ID NO: 25. Inanother example, an antibody can comprise a V_(L) comprising a sequenceas shown in SEQ ID NO: 27. In another example, the antibody comprises aV_(H) comprising a sequence as shown in any one of SEQ ID NOs: 17 to 23and a V_(L) comprising a sequence as shown in any one of SEQ ID NOs: 24to 29. For example, an antibody can comprise a V_(H) comprising asequence as shown in SEQ ID NO: 18 and a V_(L) comprising a sequence asshown in SEQ ID NO: 25. In another example, an antibody can comprise aV_(H) comprising a sequence as shown in SEQ ID NO: 21 and a V_(L)comprising a sequence as shown in SEQ ID NO: 27. In another example, anantibody can comprise a V_(H) comprising a sequence as shown in SEQ IDNO: 23 and a V_(L) comprising a sequence as shown in SEQ ID NO: 27.

In another example, an above referenced antibody can comprise a constantheavy region 1 comprising a sequence as shown in SEQ ID NO: 69. Inanother example, an above referenced antibody can comprise a constantheavy region 3 comprising a sequence as shown in SEQ ID NO: 72. Inanother example, an above referenced antibody can comprise a hingeregion comprising a sequence as shown in SEQ ID NO: 70. In theseexamples, the antibody can comprise a V_(L) comprising the amino acidsequence shown in SEQ ID NO: 27. For example, the antibody can comprisethe amino acid sequence shown in SEQ ID NO: 78.

In another example, an above referenced antibody can comprise a constantheavy region 1 comprising a sequence as shown in SEQ ID NO: 69, aconstant heavy region 3 comprising a sequence as shown in SEQ ID NO: 72,a hinge region comprising a sequence as shown in SEQ ID NO: 70 and aconstant heavy region 2 comprising a sequence as shown in any one of SEQID NOs: 71, 74, 76. In this example, the antibody can comprise a V_(L)comprising the amino acid sequence shown in SEQ ID NO: 27. For example,the antibody can comprise the amino acid sequence shown in SEQ ID NO:78.

In another example, the antibody has an amino acid sequence shown in SEQID NO: 68. In another example, the antibody has an amino acid sequenceshown in SEQ ID NO: 73. In another example, the antibody has an aminoacid sequence shown in SEQ ID NO: 75. In another example, the antibodyhas an amino acid sequence shown in any one of SEQ ID NOs: 68, 73 or 75.

As known in the art, antibodies can come in different isotypes such asIgA, IgD, IgE, IgG, and IgM. In an example, antibodies encompassed bythe present disclosure are IgG. In another example, antibodiesencompassed by the present disclosure are IgM.

In an example, the physical stability of an antibody according to thepresent disclosure is greater than an Fv such as a scFv or a di-scFvhaving corresponding V_(H) and V_(L) sequences. In an example, thephysical stability of an antibody according to the present disclosure isgreater than a di-scFv comprising an amino acid sequence as shown in SEQID NO: 50 when the antibody remains in solution at 4° C. for at leastfour weeks. In an example, the physical stability of an antibodyaccording to the present disclosure is greater than a di-scFv comprisingan amino acid sequence as shown in SEQ ID NO: 50 when the antibodyremains in solution at 4° C. for at least six months.

In another example, the antibody has reduced immunogenicity in a humansubject compared to a binding protein having a V_(H) comprising an aminoacid sequence as shown in SEQ ID NO: 48 and a V_(L) comprising an aminoacid sequence as shown in SEQ ID NO: 49. For example, an antibody canhave reduced immunogenicity compared to a binding protein having a V_(H)comprising an amino acid sequence as shown in SEQ ID NO: 48 and a V_(L)comprising an amino acid sequence as shown in SEQ ID NO: 49 whenimmunogenicity is measure via enzyme-linked immunosorbent assay (ELISA).In another example, an antibody can have reduced immunogenicity comparedto a binding protein having a V_(H) comprising an amino acid sequence asshown in SEQ ID NO: 48 and a V_(L) comprising an amino acid sequence asshown in SEQ ID NO: 49 when immunogenicity is measure via SurfacePlasmon Resonance.

In another example, the antibody has reduced immunogenicity in a humansubject compared to a di-scFv comprising an amino acid sequence as shownin SEQ ID NO: 50. For example, an antibody can have reducedimmunogenicity compared to a di-scFv comprising an amino acid sequenceas shown in SEQ ID NO: 50 when immunogenicity is measure viaenzyme-linked immunosorbent assay (ELISA). In another example, anantibody can have reduced immunogenicity compared to a di-scFvcomprising an amino acid sequence as shown in SEQ ID NO: 50 whenimmunogenicity is measure via Surface Plasmon Resonance.

In an example, the antibody has a modified Fc region. For example, theantibody Fc region can comprise an amino acid sequence as shown in SEQID NO: 71. In another example, the antibody Fc region comprises an aminoacid sequence as shown in SEQ ID NO: 74. In another example, theantibody comprises an Fc region comprising an amino acid sequence asshown in SEQ ID NO: 76. In another example, the antibody comprises an Fcregion comprising an amino acid sequence as shown in any one of SEQ IDNOs: 71, 74 or 76. In another example, the antibody comprises an Fcregion comprising an amino acid sequence as shown in SEQ ID NO: 77 withthree amino acid substitutions. In this example, the two of the aminoacid substations are between amino acid 1 and 10 of SEQ ID NO: 77. Inanother example, the two of the amino acid substations are between aminoacid 5 and 10 of SEQ ID NO: 77. In another example, the two amino acidsubstitutions are at positions 7 and 8 of SEQ ID NO: 77. In theseexamples, the third amino acid substitution is between amino acid 65 and75 of SEQ ID NO: 77. In another example, the third amino acidsubstitution is between amino acid 68 and 72 of SEQ ID NO: 77. Inanother example, the third amino acid substitution is between amino acid65 and 75 of SEQ ID NO: 77. In an example, the antibody comprises an Fcregion comprising an amino acid sequence as shown in SEQ ID NO: 77 witha L7A mutation. In another example, the antibody comprises an Fc regioncomprising an amino acid sequence as shown in SEQ ID NO: 77 with a L8Amutation. In another example, the antibody comprises an Fc regioncomprising an amino acid sequence as shown in SEQ ID NO: 77 with a N70Dmutation. In another example, the antibody comprises an Fc regioncomprising an amino acid sequence as shown in SEQ ID NO: 77 with L7A andL8A mutations. In another example, the antibody comprises an Fc regioncomprising an amino acid sequence as shown in SEQ ID NO: 77 with L7A andN70D mutations. In another example, the antibody comprises an Fc regioncomprising an amino acid sequence as shown in SEQ ID NO: 77 with L8A andN70D mutations. In another example, the antibody comprises an Fc regioncomprising an amino acid sequence as shown in SEQ ID NO: 77 with L7A,L8A and N70D mutations.

Although variation in the disclosed sequences including heavy and lightchain polypeptide sequences, and CDRs thereof, is generally providedabove with at least 95% sequence identity to the reference sequence,variants with less identity are also expressly disclosed. Thus, in someexamples, a DNA binding protein includes a polypeptide at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, or at least 94% identical to the amino acidsequence of the polypeptide of any of SEQ ID NOS: 32-47. In someembodiments, a DNA binding protein includes a variable heavy chainand/or light chain having at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, or atleast 94% identical to the amino acid sequence of the heavy and/or lightchain of any of SEQ ID NOS: 32-47 (e.g., any of SEQ ID NO:17-29). Insome embodiments, a DNA binding protein includes one or more CDRs havingleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, or at least 94% identical to theamino acid sequence of the CDRs of any of SEQ ID NOS: 32-47 (e.g., anyof SEQ ID NO: 1-16).

Binding Protein Production Recombinant Expression

In one example, a binding protein as described herein is a peptide orpolypeptide (e.g., is an antibody or antigen binding fragment thereof).In one example, the binding protein is recombinant.

In the case of a recombinant peptide or polypeptide, nucleic acidencoding same can be cloned into expression vectors, which are thentransfected into host cells, such as E. coli cells, yeast cells, insectcells, or mammalian cells, such as simian COS cells, Chinese HamsterOvary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cellsthat do not otherwise produce immunoglobulin or antibody protein.

Suitable molecular cloning techniques are known in the art anddescribed, for example in Ausubel et al., (editors), Current Protocolsin Molecular Biology, Greene Pub. Associates and Wiley-Interscience(1988, including all updates until present) or Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989). A wide variety of cloning and in vitro amplificationmethods are suitable for the construction of recombinant nucleic acids.Methods of producing recombinant antibodies are also known in the art.See U.S. Pat. No. 4,816,567 or U.S. Pat. No. 5,530,101.

Following isolation, the nucleic acid is inserted operably linked to apromoter in an expression construct or expression vector for furthercloning (amplification of the DNA) or for expression in a cell-freesystem or in cells. Thus, another example of the disclosure provides anexpression construct that comprises an isolated nucleic acid of thedisclosure and one or more additional nucleotide sequences. Suitably,the expression construct is in the form of, or comprises geneticcomponents of, a plasmid, bacteriophage, a cosmid, a yeast or bacterialartificial chromosome as are understood in the art. Expressionconstructs may be suitable for maintenance and propagation of theisolated nucleic acid in bacteria or other host cells, for manipulationby recombinant DNA technology and/or for expression of the nucleic acidor a binding protein of the disclosure.

Many vectors for expression in cells are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, a sequence encoding the binding protein(e.g., derived from the information provided herein), an enhancerelement, a promoter, and a transcription termination sequence. Exemplarysignal sequences include prokaryotic secretion signals (e.g., pelB,alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxinII), yeast secretion signals (e.g., invertase leader, α factor leader,or acid phosphatase leader) or mammalian secretion signals (e.g., herpessimplex gD signal).

Exemplary promoters active in mammalian cells include cytomegalovirusimmediate early promoter (CMV-IE), human elongation factor 1-α promoter(EF1), small nuclear RNA promoters (U1a and U1b), α-myosin heavy chainpromoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter(RSV), Adenovirus major late promoter, β-actin promoter; hybridregulatory element comprising a CMV enhancer/β-actin promoter or animmunoglobulin or antibody promoter or active fragment thereof. Examplesof useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture; babyhamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells(CHO).

Typical promoters suitable for expression in yeast cells such as forexample a yeast cell selected from the group comprising Pichia pastoris,Saccharomyces cerevisiae and S. pombe, include, but are not limited to,the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or theTEF1 promoter.

Means for introducing the isolated nucleic acid or expression constructcomprising same into a cell for expression are known to those skilled inthe art. The technique used for a given cell depends on the knownsuccessful techniques. Means for introducing recombinant DNA into cellsinclude microinjection, transfection mediated by DEAE-dextran,transfection mediated by liposomes such as by using lipofectamine(Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNAuptake, electroporation and microparticle bombardment such as by usingDNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongstothers.

The host cells used to produce the binding protein (e.g., antibody orantigen binding fragment) may be cultured in a variety of media,depending on the cell type used. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing mammalian cells. Media for culturing other celltypes discussed herein are known in the art.

The skilled artisan will understand from the foregoing description thatthe present disclosure also provides an isolated nucleic acid encoding abinding protein (e.g., a peptide or polypeptide binding protein or anantibody or antigen binding fragment thereof) of the present disclosure.

The present disclosure also provides an expression construct comprisingan isolated nucleic acid of the disclosure operably linked to apromoter. In one example, the expression construct is an expressionvector.

In one example, the expression construct of the disclosure comprises anucleic acid encoding a polypeptide (e.g., comprising a V_(H)) operablylinked to a promoter and a nucleic acid encoding another polypeptide(e.g., comprising a V_(L)) operably linked to a promoter.

The disclosure also provides a host cell comprising an expressionconstruct according to the present disclosure.

The present disclosure also provides an isolated cell expressing abinding protein of the disclosure or a recombinant cellgenetically-modified to express the binding protein.

Isolation of Proteins

Methods for purifying binding proteins according to the presentdisclosure are known in the art and/or described herein. An example isprovided in Example 1 below.

Where a peptide or polypeptide is secreted into the medium, supernatantsfrom such expression systems can be first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. A protease inhibitorsuch as PMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The binding protein prepared from cells can be purified using, forexample, ion exchange, hydroxyapatite chromatography, hydrophobicinteraction chromatography, gel electrophoresis, dialysis, affinitychromatography (e.g., protein A affinity chromatography or protein Gchromatography), or any combination of the foregoing. These methods areknown in the art and described, for example in WO99/57134 or Ed Harlowand David Lane (editors) Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, (1988).

Conjugates

In one example, a binding protein of the present disclosure isconjugated to another compound. The binding protein can be directly orindirectly bound to the compound (e.g., can comprise a linker in thecase of indirect binding). Examples of compounds include, a radioisotope(e.g., iodine-131, yttrium-90 or indium-111), a detectable label (e.g.,a fluorophore or a fluorescent nanocrystal or quantum dot), atherapeutic compound (e.g., a chemotherapeutic or an anti-inflammatory),a colloid (e.g., gold), a toxin (e.g., ricin or tetanus toxoid), anucleic acid, a peptide (e.g., a serum albumin binding peptide), aprotein (e.g., a protein comprising an antigen binding domain of anantibody or serum albumin), an agent that increases the half-life of thecompound in a subject (e.g., polyethylene glycol or other water solublepolymer having this activity) and mixtures thereof.

Methods for attaching a drug or other small molecule pharmaceutical toan antibody are well known and can include use of bifunctional chemicallinkers such as N-succinimidyl (4-iodoacetyl)-aminobenzoate;sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate;4-succinimidyl-oxycarbonyl-(2-pyridyldithio) toluene;sulfosuccinimidyl-6-[α-methyl-∀-(pyridyldithiol)-toluamido]hexanoate;N-succinimidyl-3-(-2-pyridyldithio)-proprionate;succinimidyl-6-[3(-(-2-pyridyldithio)-proprionamido]hexanoate;sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)-propionamido]hexanoate;3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent,dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine, and the like.Further bifunctional linking molecules are discussed in, for example,U.S. Pat. Nos. 5,349,066, 5,618,528, 4,569,789, 4,952,394, and5,137,877.

The linker can cleavable or noncleavable. Highly stable linkers canreduce the amount of payload that falls off in circulation, thusimproving the safety profile, and ensuring that more of the payloadarrives at the target cell. Linkers can be based on chemical motifsincluding disulfides, hydrazones or peptides (cleavable), or thioethers(noncleavable) and control the distribution and delivery of the activeagent to the target cell. Cleavable and noncleavable types of linkershave been proven to be safe in preclinical and clinical trials (see,e.g., Brentuximab vedotin which includes an enzyme-sensitive linkercleavable by cathepsin; and Trastuzumab emtansine, which includes astable, non-cleavable linker). In particular embodiments, the linker isa peptide linker cleavable by Edman degredation (Bągchor, et al.,Molecular diversity, 17 (3): 605-11 (2013)).

A non-cleavable linker can keep the active agent within the cell or thetarget microenvironment. As a result, the entire antibody, linker andactive agent enter the targeted cell where the antibody is degraded tothe level of an amino acid. The resulting complex between the amino acidof the antibody, the linker and the active agent becomes the activedrug. In contrast, cleavable linkers are catalyzed by enzymes in thetarget cell or microenvironment where it releases the active agent. Oncecleaved, the payload can escape from the targeted cell and attackneighboring cells (also referred to as “bystander killing”). In the caseof the disclosed binding proteins, cleavage of the linker can lead totwo active agents, the antibody itself and its payload, which can havedifferent mechanisms of action in the target cell or microenivornment.

In some embodiments, there is one or more additional molecules, betweenthe active agent and the cleavage site. Other considerations includesite-specific conjugation (TDCs) (Axup, Proceedings of the NationalAcademy of Sciences, 109 (40): 16101-6 (2012) and conjugation techniquessuch as those described in Lyon, et al., Bioconjugate Chem., 32 (10):1059-1062 (2014), and Kolodych, et al., Bioconjugate Chem., 26 (2):197-200 (2015) which can improve stability and therapeutic index, and αemitting immunoconjugates (Wulbrand, et al., Multhoff, Gabriele, ed.,PLoS ONE. 8 (5): e64730 (2013)).

In an example, the binding protein is conjugated to nanoparticles ormicroparticles (for example as reviewed in Kogan et al., Nanomedicine(Lond). 2: 287-306, 2007). The nanoparticles may be metallicnanoparticles. The particles can be polymeric particles, liposomes,micelles, microbubbles, and other carriers and delivery vehicles knownin the art.

If the delivery vehicle is a polymeric particle, the binding protein canbe coupled directly to the particle or to an adaptor element such as afatty acid which is incorporated into the polymer. Ligands may beattached to the surface of polymeric particles via a functional chemicalgroup (carboxylic acids, aldehydes, amines, sulfhydryls and hydroxyls)present on the surface of the particle and present on the ligand to beattached. Functionality may be introduced post-particle preparation, bycros slinking of particles and ligands with homo- or heterobifunctionalcrosslinkers. This procedure may use a suitable chemistry and a class ofcrosslinkers (CDT, EDAC, glutaraldehydes, etc. as discussed in moredetail below) or any other crosslinker that couples ligands to theparticle surface via chemical modification of the particle surface afterpreparation.

Binding proteins may also be attached to polymeric particles indirectlythough adaptor elements which interact with the polymeric particle.Adaptor elements may be attached to polymeric particles in at least twoways. The first is during the preparation of the micro- andnanoparticles, for example, by incorporation of stabilizers withfunctional chemical groups during emulsion preparation ofmicroparticles. For example, adaptor elements, such as fatty acids,hydrophobic or amphiphilic peptides and polypeptides can be insertedinto the particles during emulsion preparation. In a second embodiment,adaptor elements may be amphiphilic molecules such as fatty acids orlipids which may be passively adsorbed and adhered to the particlesurface, thereby introducing functional end groups for tethering tobinding proteins. Adaptor elements may associate with micro- andnanoparticles through a variety of interactions including, but notlimited to, hydrophobic interactions, electrostatic interactions andcovalent coupling.

Suitable polymers include ethylcellulose and other natural or syntheticcellulose derivatives. Polymers which are slowly soluble and form a gelin an aqueous environment, such as hydroxypropyl methylcellulose orpolyethylene oxide may also be suitable as materials for particles.Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA),polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA),poly-3-hydroxybut rate (PHB) and copolymers thereof,poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactoneand copolymers thereof, and combinations thereof.

Some exemplary compounds that can be conjugated to a binding protein ofthe present disclosure are listed in Table 1.

TABLE 1 Compounds useful in conjugation. Group Detail Radioisotopes¹²³I, ¹²⁵I, ¹³⁰I, ¹³³I, ¹³⁵I, ⁴⁷Sc, ⁷²As, ⁷²Sc, ⁹⁰Y, ⁸⁸Y, (eitherdirectly ⁹⁷Ru, ¹⁰⁰Pd, ^(101m)Rh, ^(101m)Rh, ¹¹⁹Sb, ¹²⁸Ba, ¹⁹⁷Hg, ²¹¹At,or indirectly) ²¹²Bi, ¹⁵³Sm, ¹⁶⁹Eu, ²¹²Pb, ¹⁰⁹Pd, ^(111n)In, ⁶⁷Gu, ⁶⁸Gu,⁶⁷Cu, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ^(99m)Tc, ¹¹C, ¹³N, ¹⁵O, ¹⁸I, ¹⁸⁸Rc, ²⁰³Pb,^(M)Cu, ¹⁰⁵Rh, ¹⁹⁸Au, ¹⁹⁹Ag or ¹⁷⁷Lu Half-life Polyethylene glycolextenders Glycerol Glucose Fluorescent Phycoerythrin (PE) probesAllophycocyanin (APC) Alexa Fluor 488 Cy5.5 Biologics fluorescentproteins such as Renilla luciferase, GFP immune modulators or proteins,such as cytokines, e.g., an interferon toxins an immunoglobulin orantibody or antibody variable region half-life extenders such as albuminor antibody variable regions or peptides that bind to albumin Chemo-Taxol therapeutics 5-FU Doxorubicin Idarubicin

In one example, a binding protein of the disclosure is conjugated to achemotherapy agent.

In one example, a binding protein of the disclosure is conjugated to amaytansinoid, e.g., DM1 or DM4.

In another example, a binding protein of the disclosure is conjugated toan auristatin, e.g., MMAE or MMAD.

In another example, a binding protein of the disclosure is conjugated toand enzyme, e.g., MTM1, GAA or AGL.

In another example, a binding protein of the disclosure is conjugated toMBNL.

In another example, a binding protein of the disclosure is conjugated toa heat shock protein (HSP). In various examples, a binding protein ofthe disclosure is conjugated to a HSP from family HSP33, HSP70, HSP90,HSP100, small HSP (sHSP) or a combination thereof. For example, abinding protein of the disclosure can be conjugated to HSP72.Accordingly, in an example, the present disclosure encompasses an Fvconjugated to a HSP from HSP70 family. In another example, the presentdisclosure encompasses an Fv conjugated to HSP72.

In another example, a binding protein of the disclosure is conjugated toa PARP inhibitor disclosed herein. For example, a binding protein of thedisclosure can be conjugated to olaparib.

In one aspect of the above examples, binding protein conjugates can beused to deliver conjugated payloads to a cell. Exemplary cells includecardiac cells such as cardiomyocytes, lung cells such as alveolar cellsand neural cells such as neurons. Other exemplary cells includecancerous cells or virally infected cells.

In some embodiments, one or more the foregoing compounds are expresslyexcluded from being conjugated to the disclosed binding proteins. Forexample, the binding protein can be naked.

Compositions

Suitably, in compositions or methods for administration of a bindingprotein according to the present disclosure to a subject, the bindingprotein is combined with a pharmaceutically acceptable carrier as isunderstood in the art. In one example, the present disclosure provides acomposition (e.g., a pharmaceutical composition) comprising a bindingprotein of the disclosure combined with a pharmaceutically acceptablecarrier. In another example, the disclosure provides a kit comprising apharmaceutically acceptable carrier suitable for combining or mixingwith a binding protein prior to administration to the subject. In thisexample, the kit may further comprise instructions for use.

In general terms, “carrier” is used to refer to a solid or liquidfiller, binder, diluent, encapsulating substance, emulsifier, wettingagent, solvent, suspending agent, coating or lubricant that may besafely administered to a subject, e.g., a human subject. Depending uponthe particular route of administration, a variety of acceptablecarriers, known in the art may be used, as for example described inRemington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA,1991).

For example, suitable carriers may be selected from a group includingsugars (e.g. sucrose, maltose, trehalose, glucose), starches, celluloseand its derivatives, malt, gelatine, talc, calcium sulfate, oilsinclusive of vegetable oils, synthetic oils and synthetic mono- ordi-glycerides, lower alcohols, polyols, alginic acid, phosphate bufferedsolutions, lubricants such as sodium or magnesium stearate, isotonicsaline and pyrogen-free water. In an example, the carrier is not H₂O.

In an example, the carrier is compatible with, or suitable for,parenteral administration. Parenteral administration includes any routeof administration that is not through the alimentary canal. Examples ofparenteral administration include injection, infusion and the like.Examples of administration by injection include intravenous,intra-arterial, intramuscular and subcutaneous injection. In anotherexample, compositions can be delivered via a depot or slow-releaseformulation which may be delivered intradermally, intramuscularly orsubcutaneously.

In some embodiments, the binding protein is encapsulated or incorporatedin nanoparticle, microparticle, or other delivery vehicle such as, butnot limited to, those discussed above.

In some embodiments, a DNA binding protein is utilized detecting site orsites of cancer, tissue damage, injury, infection, or ischemia. Themethod typically including administering to a subject in need thereof aneffective amount an agent that is detectable using diagnostic imaging ornuclear medicine techniques, and detecting the agent. In such methods,the agent is typically conjugated to the DNA binding protein orencapsulated in a delivery vehicle conjugated with the DNA bindingprotein. The diagnostic imaging or nuclear medicine technique can be,for example, PET-CT, bone scan, MRI, CT, echocardiography, ultrasound,and x-ray.

In an example, binding proteins and compositions comprising the same canbe used in the manufacture of a medicament for the treatment of acondition. In another example, the present disclosure relates to abinding protein or compositions comprising the same for use in thetreatment of a condition. Examples of conditions to be treated arediscussed below.

The methods and uses typically include administering a subject in needthere of an effective amount of a binding protein. In some embodiments,the subject has cancer or virally infected or transformed cells. In someembodiments, the subject has a disease or disorder characterized byexogenous or extracellular DNA, including but not limited to, ischemia,tissue damage, injury, or an infection. The methods and uses can includea combination therapy with a second, third, or more additional activeagents. For example, the disclosed binding proteins can be used incombination with standard chemotherapy, radiation therapy, and otheranti-cancer treatments. Radiation therapy (a.k.a. radiotherapy) is themedical use of ionizing radiation as part of cancer treatment to controlmalignant cells.

Combination Therapy

Data compiled by the present inventors indicates that the disclosedbinding proteins work with poly (ADP-ribose) polymerase (PARP)inhibitors to kill cancer cells. For example, more than additive celldeath was observed in HDR-deficient cancer cells treated with di-scFvand PARP inhibitor.

Accordingly, in another example, the present disclosure encompasses amethod of treating cancer in a subject in need thereof, the methodcomprising administering to the subject a binding protein disclosedherein and a PARP inhibitor. In another example, the present disclosurerelates to a therapeutic combination comprising a binding proteindisclosed herein and a PARP inhibitor, the combination being providedfor simultaneous or sequential administration. In another example, thepresent disclosure relates to a therapeutic combination comprising abinding protein disclosed herein and a PARP inhibitor for use intreating cancer.

In an example, the PARP inhibitor is selected from the group consistingof olaparib, niraparib, veliparib, rucaparib, talazoparib and BGB-290.For example, the PARP inhibitor can be olaparib.

Examples of binding proteins suitable for administration with a PARPinhibitor are provided above. In one example, the binding proteincomprises a V_(H) having a CDR1 as shown in SEQ ID NO: 1, a CDR2 asshown in SEQ ID NO: 2 and a CDR3 as shown in SEQ ID NO: 4 and a V_(L)having a CDR1 as shown in SEQ ID NO: 5, a CDR2 as shown in SEQ ID NO: 7and a CDR3 as shown in SEQ ID NO: 8. In another example, the bindingprotein comprises a V_(H) having a CDR1 as shown in SEQ ID NO: 1, a CDR2as shown in SEQ ID NO: 2 and a CDR3 as shown in SEQ ID NO: 4 and a V_(L)having a CDR1 as shown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7and a CDR3 as shown in SEQ ID NO: 8. In another example, the bindingprotein comprises a V_(H) having a CDR1 as shown in SEQ ID NO: 1, a CDR2as shown in SEQ ID NO: 3 and a CDR3 as shown in SEQ ID NO: 4 and a V_(L)having a CDR1 as shown in SEQ ID NO: 5, a CDR2 as shown in SEQ ID NO: 7and a CDR3 as shown in SEQ ID NO: 8. In another example, the bindingprotein comprises a V_(H) having a CDR1 as shown in SEQ ID NO: 1, a CDR2as shown in SEQ ID NO: 3 and a CDR3 as shown in SEQ ID NO: 4 and a V_(L)having a CDR1 as shown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7and a CDR3 as shown in SEQ ID NO: 8. In these examples, the bindingprotein can be an Fv. In an example, the binding protein can be adi-scFv.

Subjects having one or more of the conditions discussed below may betreated by administering a binding protein disclosed herein and a PARPinhibitor. In an example, the subject has pancreatic cancer. In anotherexample, the subject has colon cancer. In an example, the subject has acancer that is substantially BRCA2 deficient.

In another example, an above referenced combination therapy can be usedto treat subjects with cancer resistant to PARP inhibitor therapy.

In an example, the binding protein and PARP inhibitor are administeredas a single composition.

In another example, the binding protein and PARP inhibitor areadministered as separate compositions. For example, the binding proteinand PARP inhibitor can be administered simultaneously. In anotherexample, binding protein and PARP inhibitor can be administeredsequentially. In this example, administration of the binding protein andPARP inhibitor is carried out over a defined time period (usuallyminutes, hours or days). In an example, the period between sequentialadministration can be several days, provided that there is stillsufficient levels of the first therapeutic to provide or add to thetherapeutic benefit of the second therapeutic when it is administered.In one example, administration of a binding protein is followed bysequential administration of a PARP inhibitor. In another example,administration of a PARP inhibitor is followed by sequentialadministration of a binding protein.

Therapeutic combinations according to the present disclosure can beadministered via various routes. Exemplary routes of administrationinclude intravenous administration as a bolus or by continuous infusionover a period of time, intramuscular, intraperitoneal,intracerobrospinal, intrathecal, oral routes.

In an example, the binding protein and PARP inhibitor are administeredvia the same route. For example, both the binding protein and PARPinhibitor can be administered intravenously via continuous infusion. Inanother example, the binding protein and PARP inhibitor are administeredvia different routes. For example, the binding protein can administeredintravenously via continuous infusion and the PARP inhibitor can beadministered orally.

In some examples, administration of a binding protein or Fv fragmentdefined herein and a PARP inhibitor achieves a result greater than whenthe binding protein or Fv fragment and the PARP inhibitor areadministered alone or in isolation. For example, the result achieved bythe combination can be more than additive of the results achieved by theindividual components alone.

In an example, administration of the combination of a binding protein orFv fragment defined herein and a PARP inhibitor is effective to reducecancer cell proliferation or viability in a subject with cancer to agreater degree than administering to the subject the same amounts of theindividual components alone. For example, the reduction in cancer cellproliferation or viability in the subject with cancer can be more thanthe additive of the results achieved by the individual components alone.In some examples, in subjects with cancer, the combination is effectiveto reduce tumour burden, reduce tumour progression, or a combinationthereof, which may also be more than additive of the results achieved bythe individual components alone.

Conditions to be Treated

In an example, binding proteins according to the present disclosure canbe administered to a subject to treat various conditions.

In some examples of the disclosure, a method described herein is for thetreatment of a cancer. The term “cancer” refers to or describes thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemiaor lymphoid malignancies. More particular examples of such cancersinclude, but are not limited to, squamous cell cancer (e.g. , epithelialsquamous cell cancer), lung cancer including small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung and squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer including gastrointestinal cancer andgastrointestinal stromal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer ofthe urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,melanoma, superficial spreading melanoma, lentigo maligna melanoma,acral lentiginous melanomas, nodular melanomas, multiple myeloma andB-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma(NHL); mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), Meigs' syndrome,brain, as well as head and neck cancer, and associated metastases. Inanother example, the term “cancer” encompasses triple negative breastcancer. Accordingly, in an example, the present disclosure relates to amethod of treating breast, ovarian, colon, prostate, lung, brain, skin,liver, stomach, pancreatic or blood based cancer. In another example,the present disclosure relates to treating glioblastoma. In thisexample, glioblastoma may be treated by administering a binding proteindisclosed herein such as a di-scFv having SEQ ID NO: 41 or an antibodyhaving the heavy and light chain variable regions defined in SEQ ID NO:41.

In other examples, a method described herein is used to treat cancersthat are linked to mutations in BRCA1, BRCA2, PALB2, OR RAD51B, RAD51C,RAD51D or related genes. In other examples, a method described herein isused to treat cancers that are linked to mutations in genes associatedwith DNA mismatch repair, such as MSH2, MLH1, PMS2, and related genes.In other examples, a method described herein is used to treat cancerswith silenced DNA repair genes, such as BRCA1, MLH1, OR RAD51B, RAD51C,OR RAD51D.

In another example, a method described herein is used to kill cells withimpaired DNA repair processes. For example, cells with impaired DNArepair may aberrantly express a gene involved in DNA repair, DNAsynthesis, or homologous recombination. Exemplary genes include XRCC1,ADPRT (PARP-1), ADPRTL2, (PARP-2), POLYMERASE BETA, CTPS, MLH1, MSH2,FANCD2, PMS2, p53, p21, PTEN, RPA, RPA1, RPA2, RPA3, XPD, ERCC1, XPF,MMS19, RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCCR, XRCC3, BRCA1, BRCA2,PALB2, RAD52, RAD54, RAD50, MREU, NB51, WRN, BLM, KU70, KU80, ATM, ATRCPIK1, CHK2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG,FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, and RADS. In anexample, a method described herein can be used to kill HDR deficientcells. In another example, a method described herein is used to killcells with a mutant tumor suppressor gene. For example, cells can haveone or more mutations in BRCA1 or BRCA2. For example, cells can be BRCA2deficient colon cancer cells.

In an example, a method described herein is for the treatment of acancer that is substantially HDR deficient. In an example, a methoddescribed herein is for the treatment of a cancer that is substantiallyBRCA2 deficient. For example, a BRCA2 deficient colon cancer may betreated. In another example, a method described herein is for thetreatment of a cancer that is substantially PTEN deficient. For example,a PTEN deficient brain cancer may be treated. In another example, amethod described herein is for the treatment of a cancer that isresistant to PARP inhibition.

In other examples of the disclosure, a method described herein is usedto treat virally transformed cells, such as cells infected with anoncovirus. The term “oncovirus” is used in the context of the presentdisclosure to refer to viruses that are able to replicate in and reducegrowth of tumour cells. In an example, the oncovirus virus is able tonaturally replicate in and reduce growth of tumour cells. Examples ofsuch viruses include Newcastle disease virus, vesicular stomatitis,myxoma, reovirus, sindbis, measles and coxsackievirus. In anotherexample, the oncovirus virus is engineered to replicate in and reducegrowth of tumour cells. Exemplary viruses suitable for such engineeringinclude adenovirus, herpes simplex virus (HSV), lentivirus, vaccina andvesicular stomatitis virus (VSV).

Other exemplary oncoviruses include Human papillomaviruses (HPV),Hepatitis B (HBV), Hepatitis C (HCV), Human T-lymphotropic virus (HTLV),Kaposi's sarcoma-associated herpesvirus (HHV-8), Merkel cellpolyomavirus, Epstein-Barr virus (EBV), Human immunodeficiency virus(HIV), and Human cytomegalovirus (CMV).

In other examples of the disclosure, a method described herein is usedto kill cells transformed with a latent virus. Exemplary latent virusesinclude CMV, EBV, Herpes simplex virus (type 1 and 2), and Varicellazoster virus.

In other examples of the disclosure, a method described herein is usedto treat active viral infections due to viruses that give rise tocancer, immunodeficiency, hepatitis, encephalitis, pneumonitis orrespiratory illness. Exemplary viruses include above referencedoncovirus, parvovirus, poxvirus, herpes virus.

In other examples of the disclosure, a method described herein is usedto treat Colorado Tick Fever (caused by Coltivirus, RNA virus), WestNile Fever (encephalitis, caused by a flavivirus that primarily occursin the Middle East and Africa), Yellow Fever, Rabies (caused by a numberof different strains of neurotropic viruses of the familyRhabdoviridae), viral hepatitis, gastroenteritis (viral)-acute viralgastroenteritis caused by Norwalk and Norwalk-like viruses, rotaviruses,caliciviruses, and astroviruses, poliomyelitis, influenza (flu), causedby orthomyxoviruses that can undergo frequent antigenic variation,measles (rubeola), paramyxoviridae, mumps, respiratory syndromesincluding viral pneumonia and acute respiratory syndromes includingcroup caused by a variety of viruses collectively referred to as acuterespiratory viruses, and respiratory illness caused by the respiratorysyncytial virus.

In other examples of the disclosure, a method described herein is usedto treat a nucleotide repeat disorder or an exon splicing disorder. Inother examples of the disclosure, a method described herein is used totreat a disorder associated with aberrant microsatellite expansion, suchas myotonic dystrophy. For example, the methods of the presentdisclosure may be used to treat Myotonic dystrophy. Examples of Myotonicdystrophy type 1 (DM1; trinucleotide (CTG)_(n) expansion of n=50to >3000 in the 3′-untranslated region of the Dystrophiamyotonica-protein kinase (DMPK) gene) and type 2 (DM2; tetranucleotide(CCTG)_(n) expansion of n=75 to about 11,000 in the first intron of zincfinger protein 9 (ZNF9) gene. In other examples of the disclosure, amethod described herein is used to treat neurofibramotosis. In otherexamples of the disclosure, a method described herein is used to treatHuntington's Disease. In other examples of the disclosure, a methoddescribed herein is used to treat myotubular myopathy. In other examplesof the disclosure, a method described herein is used to treat a glycogenstorage disorder. In other examples of the disclosure, a methoddescribed herein is used to treat Pompe Disease. In other examples ofthe disclosure, a method described herein is used to treat Forbes-CoriDisease. In other examples of the disclosure, a method described hereinis used to treat Lafora Disease.

In other examples of the disclosure, a method described herein is usedto increase Muscleblind-like (MBNL) activity in a cell in vitro or in asubject by administering a binding protein according to the presentdisclosure conjugated to an MBNL polypeptide. In other examples of thedisclosure, a method described herein is used for enzyme or proteinreplacement therapy.

In other examples of the disclosure, a method described herein is usedto increase HSP activity in a cell in vitro or in a subject byadministering a binding protein according to the present disclosureconjugated to a HSP from HSP70 family. In other examples of thedisclosure, a method described herein is used to increase HSP72 activityin a cell in vitro or in a subject by administering a binding proteinaccording to the present disclosure conjugated to an HSP72 polypeptide.

EXAMPLES Example1—Expression and Purification of di-scFV Variants

Single gene GS vectors (using Lonza's GS Xceed™ Gene Expression System)were established, sequenced, linearized and used to generate a stablepool for each variant. Following cryopreservation the propagated stablepools were expanded to 200 mL culture volume each and subjected to anabridged fed batch overgrow with a single bolus feed on day 4 andharvested on day 8. Supernatant titre was determined by Protein L Octet.Clarified supernatant for ion exchange purification was obtained bycentrifugation followed by filter sterilisation using a 0.22 μm filter.An ion exchange purification method was developed using the dimerversion of the murine antibody as a reference.

Clarified supernatant was purified using a pre-packed 5 mL HiTrap CaptoS column (GE Healthcare, 17-544122) on an AKTA purifier (run at 5mL/min). The column was equilibrated with 50 mM Sodium Phosphate pH 6before and after sample loading and the product was eluted with a lineargradient from 0-1 M NaCl. Quantification of bound and unbound materialby Protein L Octet showed that approximately 57% of material remained inthe unbound fraction. Repeating the chromatography using the unboundfraction again resulted in approximately 64% of the starting materialremaining in the unbound fraction.

Purification of the remaining supernatants was performed using twosequential steps of ion exchange chromatography with a linear elutiongradient from 0-1 M NaCl. Following purification, the products werequantified and concentrated to approximately 1 mg/mL by ultrafiltrationusing Amicon Ultra-15 filters (Millipore, UFC903024).

Duplicate samples were analysed by SE-HPLC on an Agilent 1200 seriesHPLC system, using a Zorbax GF-250 9.4 mm ID×25 cm column (Agilent) andby SDS-PAGE analysis. Yields and titres of expression cultures aresummarised in Table 1. SDS-PAGE analysis of variants is shown in FIGS. 1and 2.

TABLE 1 Yields and titres of expression cultures. Estimated Final Vol-Final Mono- Titre concentration ume Yeild mer Product (mg/L) (mg/mL)(mL) (mg) (%) var_2 393.4 1.079 3.2 3.5 84.08 var_3 436.8 1.156 1.5 1.780.61 var_4 445.0 1.090 2.5 2.7 84.26 var_6 275.7 1.214 1.6 1.9 93.02var_7 288.4 0.829 1.5 1.2 79.16 var_8 373.7 1.024 2.0 2.0 81.71 var_10325.2 0.767 5.6 4.3 85.17 var_11 349.7 1.181 6.2 7.3 81.86 var_12 396.11.169 4.0 4.7 80.86 var_13 459.1 0.803 5.0 4.0 86.13 var_14 527.5 0.7994.0 3.2 82.72 var_15 584.2 1.003 3.2 3.2 86.34 var_16 391.9 0.842 5.64.7 85.53 var_17 315.6 1.106 1.8 2.0 85.79 var_18 460.3 1.118 4.5 5.085.37 var_19 318.9 0.401 3.1 1.2 84.47 tri_L1H2 251.4 1.091 3.2 3.595.47 Di_scFv_B72.3 55.7 0.0 0.0 di_scFv_D31N 270.7 1.027 6.6 6.8 95.36tri_scFv_D31N 40.2 0.658 2.5 1.6 93.34

Example 2—Nuclear Penetration of Variants Alkaline Phosphatase-BasedSurvey of Nuclear Penetration

DLD1 colon cancer cells were treated with control media or each of theindicated variants for one hour. Cells were then washed, fixed, blockedwith 1% BSA-TBST, and then probed with protein L for one hour. Cellswere then washed and incubated with an anti-protein L primary antibodyfor one hour. After another round of washing cells were incubated withan alkaline phosphatase-conjugated secondary antibody for one hour.Finally, cells were washed and signal was developed by addition ofNBT/BCIP. Representative images are shown in FIG. 3. Dark stainindicates location of the variants.

Raw integrated density values reflecting nuclear alkaline phosphatasestaining in the DLD1 cells from the experiment in FIG. 3 were obtainedby analysis using ImageJ. Boxplots of distributions of values arepresented for each variant in FIG. 4.

Histogram plots of cell counts versus nuclear staining intensity(represented as reciprocal intensity in arbitrary units) are shown inFIG. 5. Most of the variants, other than variants 12 and 14, showedimproved nuclear penetration relative to the yeast prototype, which isdemonstrated by right shift of histogram peak. In addition, thenarrowing of distributions observed in the histograms for most of thehumanized variants shows improved uniformity of nuclear penetrationrelative to the yeast prototype. Variants 13 and 15 in particular showednotable right shift and narrowing of distributions relative to the yeastprototype.

Immunofluorescence-Based Survey of Nuclear Penetration

DLD1 colon cancer cells were treated with control media or each of theindicated variants for one hour. Cells were then washed, fixed, blockedwith 1% BSA-TBST, and then probed with protein L for one hour. Cellswere then washed and incubated with an anti-protein L primary antibodyfor one hour. After another round of washing cells were incubated withan Alexa488-conjugated secondary antibody for one hour. Finally, cellswere washed and signal was visualized by fluorescence microscopy.Representative images are shown in FIG. 6. Green signal indicateslocation of the variants.

Raw integrated density values reflecting Alexa488 fluorescence signal inthe DLD1 cells from the experiment in FIG. 6 were obtained by analysisusing ImageJ. Boxplots of distributions of values are presented for eachvariant in FIG. 7.

Example 2—Accumulation of DNA Damage

A matched pair of PTEN-proficient and deficient U251 human glioma cellswere treated with control media or media containing Variant 10, 11, 13,15, or 16 for twenty-four hours. Cells were then washed, fixed, blocked,and then probed with an anti-phospho-53BP1 antibody overnight. Cellswere then washed and incubated with an AlexaFluor555-conjugatedsecondary antibody. Finally, cells were washed, counterstained withDAPI, and visualized under a fluorescence microscope. Images were savedand evaluated by CellProfiler to determine mean number of phospho-53BP1foci per cell. The new variants increased the number of foci in thePTEN-deficient cells, but not the PTEN-proficient cells. Representativeimages are shown in FIG. 8, Panel A, and quantitative analysis byCellProfiler is shown in FIG. 8, Panel B.

Cell viability of PTEN-deficient U87 human glioma cells was alsoassessed following treatment with control media or media containingVariants 10, 13, 15, or 16. Cell viability was determined 7 days aftertreatment using Trypan blue exclusion assay and by direct visualizationof cell morphology by light microscopy. All variants caused reductionsin cell viability relative to control treated cells (FIG. 9).

Next, a matched pair of BRCA2-proficient and deficient DLD1 colon cancercells was treated with control media or media containing Variants 10,13, 15, or 16. Cell viability was determined 7 days later by Trypan blueexclusion assay and by direct visualization of cell morphology by lightmicroscopy. The variants were not toxic to the BRCA2-proficient cells,but the BRCA2-deficient cells were killed by the variants. These dataindicate that variants are able to selectively kill cancer cells withimpaired DNA repair. Moreover, these data indicate that that thevariants will be able to discriminate between cancerous cells withimpaired DNA repair and healthy cells to selectively kill cancer cells.Representative light microscope images shown the changes in morphologyin the BRCA2-deficient cancer cells treated with the variants are shownin FIG. 10, Panel A. Quantitative analysis of the cell survival byTrypan blue exclusion assay is shown in FIG. 10, Panel B.

Example 3—Di-scFv Co-Administration with PARP Inhibition in HDRDeficient Cancer Cells

DLD-1 and MCF-7 cells were treated with control or di-scFv (SEQ ID NO:41), and nuclear penetration was evaluated by protein L immunostain offixed cells. The di-scFv successfully penetrated DLD-1 and MCF-7 cellnuclei (FIGS. 11 and 12).

Homology-directed repair (HDR) deficient BRCA2- DLD1 cells and PTEN-U251cells were treated with control, 5 nM olaparib, 10 μM di-scFv, or 10 μMdi-scFv+5 nM olaparib. Surviving fraction was determined by colonyformation assay. Surprisingly, more than additive cell death wasobserved in HDR-deficient cancer cells treated with di-scFv and the PARPinhibitor (FIG. 13).

It was then determined whether the combination of di-scFv and olaparibis simply universally cytotoxic, regardless of DNA repair status. Toevaluate this possibility, HDR-proficient DLD1 cells were treated withthe above regimen to confirm selectivity of combination therapy toHDR-deficient malignant cells. No effect on cell death was observed forthe di-scFv alone or in combination with PARP inhibitor. These findingsdemonstrate that HDR-proficient cells remain resistant to the effects ofboth the di-scFv and olaparib, even when used in combination.

Example 4—Effect of Di-scFv (SEQ ID NO: 41) on Primary HumanGlioblastoma (GBM) Cells

Primary human glioblastoma (GBM) cancer cells extracted from primaryhuman GBM tumours from patients were treated with control or di-scFv(SEQ ID NO: 41), and percentage of live cells was evaluated by trypanblue staining. Five of the seven glioblastoma tumour explants treatedwith di-scFv (SEQ ID NO: 41) showed significant cancer cell death (FIG.14).

GBM cancer stem cells extracted from primary human GBM tumours frompatients and grown as spheres were treated with control or di-scFv (SEQID NO: 41), and the effect of dose and incubation time on reduction ofsphere volume was evaluated by confocal micrographs of DX1-rhodaminecellular penetration into GBM cells. Tumour spheres are recognised as auseful tool for pre-clinical studies as they retain tumour heterogeneityand more closely represent the original patient tumour. Treatment ofhuman GBM cancer stem cells (CSCs) grown as tumour spheres with di-scFv(SEQ ID NO: 41) demonstrated cellular penetration in GBM spheres andreduced sphere volume in dose-dependent and time-dependant manner (FIG.15).

Example 5—Evaluation of the Effect of di-scFv (SEQ ID NO: 41) on HumanGBM Cells in an Orthotopic Mouse Model

An orthotopic mouse model of GBM was generated by intracranial injectionof GBM cells extracted from human GBM tumours. Once the tumoursdeveloped in the brain, mice were treated by tail vein injection ofcontrol or di-scFv variant 13 (SEQ ID NO: 41), and effect of di-scFv onreduction of tumour volume was evaluated by extraction of tumours.Evaluation of brain sections showed that the glioblastoma tumours inmice treated with di-scFv were more than 40% smaller than the comparabletumours in control mice (FIG. 16A). TUNEL staining also demonstratedincreased incidence of apoptosis in di-scFv-treated tumours (FIG. 16B).The observed reduction in tumour size and increased TUNEL staining inthe di-scFv-treated GBM tumours suggested that di-scFv variant 13 (SEQID NO: 41) successfully crossed the blood brain barrier to localize inand impact GBM tumour growth. To confirm this, tumours and normal brainwere probed for di-scFv by protein L immunostaining. As shown in FIG.16C, the di-scFv was detected in the nuclei of GBM tumour cells, but wasnot evident in surrounding adjacent normal brain cells.

Additionally, a group of 7 mice was evaluated for the survival benefitand mice treated with di-scFv showed a median survival of 87 days, morethan 20% longer than controls (median 72 days). Mean survival datareflected these trends (83 days±3.2 days for di-scFv treated mice, 71days±1.2 days for controls) (FIG. 16E). Statistical analysis indicated asignificant difference between the two groups, with P value=0.004. Notoxicity or weight loss associated with di-scFv treatments was observed(FIG. 16D).

Example 6—The Effect of PAT-DX1 on Foci Accumulation

A matched pair of BRCA2-proficient and deficient DLD1 colon cancer cellsand PTEN-proficient and deficient U251 human glioma cells were treatedwith control media or media containing 10 μM di-scFv variant 13 (SEQ IDNO: 41), 5 nM olaparib, or combination treatment. Phospho-53BP1 antibodystaining was evaluated by Cell Profiler to determine mean number ofphospho-53BP1 foci per cell. di-scFv variant 13 (SEQ ID NO: 41)treatment alone and in combination with olaparib increased the number ofphospho-53BP1 foci in both the BRCA2-deficient DLD1 and thePTEN-deficient U251 cells, but not in proficient cells (FIG. 17).

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the disclosure as shownin the specific embodiments without departing from the spirit or scopeof the disclosure as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed above are incorporated herein in theirentirety. Any discussion of documents, acts, materials, devices,articles or the like which has been included in the presentspecification is solely for the purpose of providing a context for thepresent disclosure. It is not to be taken as an admission that any orall of these matters form part of the prior art base or were commongeneral knowledge in the field relevant to the present disclosure as itexisted before the priority date of each claim of this application.

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1. A cell penetrating anti-DNA binding protein having an antigen bindingdomain, wherein the antigen binding domain binds to DNA and comprises: aheavy chain variable region (V_(H)) having a complementarity determiningregion (CDR) 1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2or SEQ ID NO: 3 and a CDR3 as shown in SEQ ID NO: 4; a light chainvariable region (V_(L)) having a CDR1 as shown in SEQ ID NO: 5 or SEQ IDNO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ ID NO:8.
 2. A cell penetrating anti-DNA binding protein having an antigenbinding domain, wherein the antigen binding domain binds to DNA andcomprises: a heavy chain variable region (V_(H)) having acomplementarity determining region (CDR) 1 as shown in SEQ ID NO: 9, aCDR2 as shown in SEQ ID NO: 10 or SEQ ID NO: 11 and a CDR3 as shown inSEQ ID NO: 12; a light chain variable region (V_(L)) having a CDR1 asshown in SEQ ID NO: 13 or SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO:15 and a CDR3 as shown in SEQ ID NO:
 16. 3. The binding protein of claim1 or claim 2 comprising: (i) a V_(H) comprising a sequence at least 95%identical to the sequence as shown in any one of SEQ ID NOs: 17 to 23;(ii) a V_(L) comprising a sequence at least 95% identical to thesequence as shown in any one of SEQ ID NOs: 24 to 29; or (iii) a V_(H)comprising a sequence at least 95% identical to the sequence as shown inany one of SEQ ID NOs: 17 to 23 and a V_(L) comprising a sequence atleast 95% identical to the sequence as shown in any one of SEQ ID NOs:24 to
 29. 4. The binding protein according to any one of claims 1 to 3,wherein the V_(H) and a V_(L) are separated by a linker.
 5. The bindingprotein of claim 4, wherein the linker comprises the sequence shown inSEQ ID NO:
 30. 6. The binding protein of any one of claims 1 to 5,wherein, the V_(H) and V_(L) are in a single polypeptide chain.
 7. Thebinding protein of claim 6, which is: (i) a single chain Fv fragment(scFv); (ii) a dimeric scFv (di-scFv); (iii) a trimeric scFv (tri-scFv);(iv) any one of (i), (ii) or (iii) linked to a constant region of anantibody, Fc or a heavy chain constant domain C_(H)2 and/or C_(H)3. 8.The binding protein of claim 6, which is a scFv.
 9. The binding proteinof claim 6, which is a di-scFv.
 10. The binding protein of claim 9,wherein the scFv' s are separated by a linker.
 11. The binging proteinof claim 10, wherein the linker comprises the sequence shown in SEQ IDNO:
 31. 12. The binding protein according to any one of claims 1 to 5,wherein, the V_(H) and V_(L) are in a separate polypeptide chain. 13.The binding protein of claim 12, which is: (i) a diabody; (ii) atriabody; (iii) a tetrabody; (iv) a Fab; (v) a F(ab′)₂; (vi) a Fv; (vii)one of (i) to (vi) linked to a constant region of an antibody, Fc or aheavy chain constant domain C_(H)2 and/or C_(H)3; or, (viii) an intactantibody.
 14. A cell penetrating anti-DNA Fv fragment having an antigenbinding domain, wherein the antigen binding domain binds to DNA andcomprises at least one of: a V_(H) having a CDR 1 as shown in SEQ ID NO:1, a CDR2 as shown in SEQ ID NO: 2 or SEQ ID NO: 3, a CDR3 as shown inSEQ ID NO: 4 and a V_(L) having a CDR1 as shown in SEQ ID NO: 5 or SEQID NO: 6, a CDR2 as shown in SEQ ID NO: 7 and a CDR3 as shown in SEQ IDNO: 8, a V_(H) having a CDR 1 as shown in SEQ ID NO: 9, a CDR2 as shownin SEQ ID NO: 10 or SEQ ID NO: 11, a CDR3 as shown in SEQ ID NO: 12 anda V_(L) having a CDR1 as shown in SEQ ID NO: 13 or SEQ ID NO: 14, a CDR2as shown in SEQ ID NO: 15 and a CDR3 as shown in SEQ ID NO: 16; a V_(H)comprising a sequence at least 95% identical to the sequence as shown inany one of SEQ ID NOs: 17 to 23 and a V_(L) comprising a sequence atleast 95% identical to the sequence as shown in any one of SEQ ID NOs:24 to
 29. 15. The cell penetrating anti-DNA Fv fragment of claim 14which is a di-scFv.
 16. The cell penetrating anti-DNA Fv fragment ofclaim 15, which comprises an amino acid sequence as shown in any one ofSEQ ID NOs: 32-47.
 17. The binding protein according to any one ofclaims 1 to 13 or the Fv fragment according to any one of claims 14-16,which is conjugated to another compound.
 18. A nucleic acid encoding abinding protein or Fv fragment defined by any one of claims 1 to
 17. 19.An expression construct comprising a nucleic acid defined by claim 18.20. An isolated or recombinant cell expressing a binding protein or Fvfragment defined by any one of claims 1 to 17, a nucleic acid defined byclaim 18 or the expression vector of claim
 20. 21. A compositioncomprising a binding protein or Fv fragment defined by any one of claims1 to 17 and a pharmaceutically acceptable carrier.
 22. A method oftreating cancer in a subject, the method comprising administering to thesubject and effective amount of a binding protein or Fv fragment definedby any one of claims 1 to 17 or the composition of claim
 21. 23. Themethod claim 22, wherein the cancer is glioblastoma.
 24. Use of abinding protein or Fv fragment defined by any one of claims 1 to 17 orthe composition of claim 21 in the manufacture of a medicament fortreating cancer.
 25. A binding protein or Fv fragment defined by any oneof claims 1 to 17 or the composition of claim 21 for use in treatingcancer.
 26. A method of treating cancer in a subject in need thereof,the method comprising administering to the subject a binding protein orFv fragment defined by any one of claims 1 to 17 and a PARP inhibitor.27. The method of claim 26, wherein the PARP inhibitor is olaparib. 28.The method according to claim 26 or claim 27, wherein the cancer issubstantially HDR deficient.
 29. The method according to any one ofclaims 26 to 28, wherein the cancer is resistant to PARP inhibition. 30.The method according to any one of claims 26 to 29, wherein the canceris substantially BRCA2 deficient.
 31. The method according to any one ofclaims 26 to 29, wherein the cancer is substantially PTEN deficient. 32.The method according to any one of claims 26 to 31, wherein the canceris colon cancer, brain cancer, prostate, ovarian, breast, endometrial,melanoma, or pancreatic cancer.
 33. The method according to any one ofclaims 26 to 31, wherein the cancer is a triple negative breast cancer.34. The method according to any one of claims 26 to 31, wherein thecancer is a glioblastoma.